qPCR 2011 proceedings

Transcription

qPCR 2011.net 28th March - 1st April 2011
Symposium & Exhibition & Workshops
Molecular Diagnostics:
from single-cells to Next Generation Sequencing
5th international qPCR Event, TUM, Freising-Weihenstephan, Germany
qPCR 2011 Event Proceedings
(online version)
5th International qPCR Symposium
Industrial Exhibition & Application Workshops
Molecular Diagnostics:
from single-cells to Next Generation Sequencing
Editor: Michael W. Pfaffl
Physiology, Freising – Weihenstephan
Weihenstephaner Berg 3
Technical University Munich (TUM)
85354 Freising
Germany
www.qPCR2011.net
ISBN 9783000338403
qPCR 2011 – Online Proceedings – page 2
qPCR 2011 Sponsors
Lead Sponsors:
Gold Sponsors:
Silver Sponsors:
Further Companies in the Industrial Exhibition:
Media partners:
Table of content
qPCR 2011 Sponsors................................................................................................................................................................... 2
Table of content ........................................................................................................................................................................... 3
Invitation ....................................................................................................................................................................................... 4
qPCR 2011 Event Agenda............................................................................................................................................................ 5
Sunday 27th March 2011........................................................................................................................................................ 6
Agenda - Monday 28th March 2011 ........................................................................................................................................... 6
Welcome & Opening of the Symposium.............................................................................................................................. 6
MIQE and QC strategies in qPCR ........................................................................................................................................ 6
single-cell qPCR ................................................................................................................................................................... 6
Agenda - Tuesday 29th March 2011........................................................................................................................................... 7
High throughput & digital PCR.............................................................................................................................................. 7
NGS: Next Generation Sequencing ...................................................................................................................................... 7
RNAi - microRNA - siRNA Applications ................................................................................................................................ 8
qbasePLUS: data analysis lunch seminar ............................................................................................................................ 8
New qPCR Applications and Method Optimisation............................................................................................................... 8
th
Agenda - Wednesday 30 March 2011 ....................................................................................................................................... 9
Pre-PCR: Pre-analytical Steps ............................................................................................................................................. 9
New qPCR Applications in Molecular Diagnostics ................................................................................................................ 9
Data Analysis: qPCR BioStatistics & BioInformatics........................................................................................................... 10
GENEX: data analysis lunch seminar ................................................................................................................................. 10
MIQE: state of the art & open discussion........................................................................................................................... 10
RDML: state of the art & open discussion.......................................................................................................................... 10
Closing of the Symposium ................................................................................................................................................... 10
st
st
Agenda - Thursday 31 March & Friday 1 April 2011.......................................................................................................... 11
qPCR Application Workshops.............................................................................................................................................. 11
Basic Module qPCR Application Workshop (2-days)………………… ............................................................................... 11
qPCR data analysis - Biostatistics & Expression Profiling (2-days)……………………....................................................... 11
MIQE guidelines (1 day) & Practical primer design (1-day) ……………………............................................................... 12
qPCR Workshop Sponsors:................................................................................................................................................ 12
Industrial Exhibition................................................................................................................................................................... 13
Abstracts - Oral presentations.................................................................................................................................................. 14
th
Monday 9 March 2009 ........................................................................................................................................................ 14
MIQE and QC strategies in qPCR ...................................................................................................................................... 14
single-cell qPCR ................................................................................................................................................................. 15
Tuesday 29th March 2011 .................................................................................................................................................... 17
High throughput & digital PCR............................................................................................................................................ 17
NGS: Next Generation Sequencing .................................................................................................................................... 18
RNAi - microRNA - siRNA Applications .............................................................................................................................. 20
qbasePLUS: data analysis lunch seminar .......................................................................................................................... 21
New qPCR Applications and Method Optimisation............................................................................................................. 21
th
Wednesday 30 March 2011................................................................................................................................................. 23
Pre-PCR: Pre-analytical Steps ........................................................................................................................................... 23
New qPCR Applications in Molecular Diagnostics .............................................................................................................. 25
Data Analysis: qPCR BioStatistics & BioInformatics........................................................................................................... 27
GENEX: data analysis lunch seminar ................................................................................................................................. 29
MIQE: state of the art & open discussion........................................................................................................................... 29
RDML: state of the art & open discussion.......................................................................................................................... 29
Abstracts - Poster presentations.............................................................................................................................................. 30
New qPCR Applications: Molecular Diagnostics & Expression Profiling P001 – P046 ..................................................... 30
New qPCR Applications: Method Optimisation & Standardisation P047 – P055 .............................................................. 42
qPCR Biostatistics & Bioinformatics P056 – P061............................................................................................................ 45
High Throughput qPCR & digital PCR Next Genartion Sequencing P062 – P069............................................................ 46
Single-cell qPCR qPCR from limited material P070 – P075 ............................................................................................. 48
HRM & Methylation Studies P076 – P084 ........................................................................................................................ 50
RNAi: microRNA – siRNA Applications P085 – P090 ...................................................................................................... 53
List of participants ..................................................................................................................................................................... 55
Invitation
Dear colleagues,
Dear researchers,
Dear company representatives,
On behalf of the organisation committee and the scientific board of the conference it is a great pleasure to invite you to the
qPCR 2011 Event, the 5
th
International qPCR Symposium, including an Industrial Exhibition and three qPCR Application
Workshops. The symposium focus is on Molecular Diagnostics: from single-cells to Next Generation Sequencing. 71
lectures and 91 posters will be presented by international recognised experts in their application fields. The emphasis will be on
unbiased, didactic information exchange. One third of the talks will be held by invited speakers, one third of the speakers will be
selected from the submitted abstracts and one third of the oral contributions will be done by qPCR company representatives.
The poster sessions will be shown on Monday evening and around lunch time on Tuesday and Wednesday in the poster
exhibition hall below the foyer.
The qPCR Event is structured in three parts:
1.
qPCR Symposium taking place March 28-30, including various talk and poster sessions
2.
A parallel qPCR Industrial Exhibition with 35 company booth taking place March 28-30
3.
Followed by three qPCR Workshops taking place March 31 – April 1st
powered by BioEPS GmbH and the TATAA Biocenter Sweden
The scientific organization is managed by international well-known scientists in the field of real-time PCR:
Stephen Bustin
Prof. of Molecular Science, School of Medicine, London, UK
Mikael Kubista
Prof. of Biotechnology, TATAA Biocenter, Sweden
Jo Vandesompele
Prof. at the Center of Medical Genetics, University of Ghent, Belgium
Heinrich H. D. Meyer
Prof. of Physiology, Technical University of Munich, Weihenstephan, Germany
Michael W. Pfaffl
Prof. of Molecular Physiology, TUM Weihenstephan, Germany (scientific coordination)
Event organization will be supervised by Dr. Martina Reiter, BioEPS GmbH
[email protected]
The qPCR 2011 event location is the central lecture hall complex and the foyer at TUM (Technical University of Munich) in
Freising Weihenstephan, Germany. The TUM and the Biotech region around Munich are part of the largest Biotech cluster in
Europe, located close to the Munich airport (MUC) directly in the heart of Bavaria.
Please enjoy the conference, the exhibition, the workshops and the social program during the following week.
Best regards
scientific coordinator
qPCR 2011 Event Agenda Overview
online agenda (HTML version) => http://agenda.qPCR2011.net
Lecture hall 14 (HS 14)
online agenda (PDF version) => http://agendaPDF.qPCR2011.net
Lecture hall 15 (HS 15)
12:00 – 18:00
Industrial Exhibition Built up
Sunday
27th Mach
15:00 – 18:00
Arrival & Registration
10:00 – 10:30
Monday
28th March
Foyer &
Seminar rooms S1 & S2
8:00 – 10:00
Welcome & Opening
qPCR 2011 Symposium
8:00 – 10:00
Poster Setup
in Foyer lower level
Welcome by Michael W. Pfaffl &
Alfons Gierl Dean WZW TUM
10:30 – 13:00
MIQE & QC strategies in qPCR
10:00 – 21:00
Industrial Exhibition
13:00 – 14:00
Lunch
14:00 – 18:00
Single-cell qPCR
18:00 – 21:00 Reception in the Industrial Exhibition
8:30 – 12:00
High Throughput qPCR
& digital PCR
Tuesday
8:30 – 12:00
RNAi:
microRNA – siRNA Applications
12:00 – 14:00
Lunch
29th March
13:00 – 14:00
qbasePLUS
data analysis lunch seminar
14:00 – 18:00
New qPCR Applications
& Method Optimisation
14:00 – 18:00
Next Generation Sequencing
19:00 – 24:00
18:00 – 21:00
Evening Poster Session
8:30 – 14:00
12:00 – 14:00
Lunch Poster Session
14:00 – 18:00
Symposium Gala Dinner
Location: Lindenkeller, Pasta & More, Freising
International Buffet, Asian Buffet, Music & Dancing
9:00 – 12:00
Pre-analytical Steps
Wednesday
9:00 – 12:00
Biostatistics & Bioinformatics
12:00 – 14:00
Lunch
30th March
13:45 – 16:30
New qPCR Applications
in Molecular Diagnostics
13:00 – 14:00
GenEx
data analysis lunch seminar
14:00 – 15:00
MIQE
state of the art & open discussion
15:30 – 16:30
RDML
state of the art & open discussion
16:30 – 17:00
Closing of the Symposium
Heinrich HD. Meyer & Michael W. Pfaffl
Thursday
31st March
Friday
1st April
9:00 – 16:30
9:00 - 17:00
qPCR Workshops:
- Basis Module qPCR Application Workshop (2-days)
- qPCR data analysis: Biostatistics & Expression Profiling (2-days)
- MIQE guidelines (1 day) & Practical primer design (1-day)
12:00 – 14:00
14:00 – 16:30
Poster Take Down
Agenda qPCR 2011 Event
12:45
Sunday 27th March 2011
12:00 – 18:00
Built-up for Industrial Exhibition
15:00 – 18:00
Monday 28th March 2011
Welcome & Opening of the Symposium
Lecture hall HS 14
08:00 – 10:00
Built-up for Industrial Exhibition
09:00 – 10:00
Welcome Coffee & Tea
Substantial performance discrepancies between
commercially available kits for rt-qPCR - A systematic
comparative investigator-driven approach
Matthias W Sieber1,2, Peter Recknagel2, Florian Glaser2,
Otto W. Witte1, Michael Bauer2, Ralf A. Claus2, Christiane
Frahm1
1
Hans Berger Clinic for Neurology, Jena University
Hospital, Germany; 2Anaesthesiology and Intensive Care
Medicine, Jena University Hospital, Germany
13:00 – 14:00
Lunch in the student cafeteria
single-cell qPCR
Time:
Location:
Session Chair:
Monday, 28/03/2011: 2:00pm - 6:00pm
Lecture hall 14
Mikael Kubista, Anders Ståhlberg
10:00
Welcome & Opening of the Symposium
Michael W. Pfaffl
Scientific coordination of the qPCR 2009 Symposium
14:00
10:15
Welcome at the Center of Food & Life Science in
Freising Weihenstephan
Alfons Gierl
Dean WZW Technical University of Munich
25 years of PCR - from idea to subcellular expression
profiling
Mikael Kubista
TATAA BIOCENTER, Sweden
14:30
Uncovering the Diversity of Individual Cells: Gene
Expression Profiling with the BioMark System
Ken Livak
Fluidigm Corporation, United States of America
15:00
Identification of unknown cell populations and
correlations using single-cell gene expression
profiling
Anders Ståhlberg
University of Gothenburg, Sweden
MIQE and QC strategies in qPCR
Time:
Location:
Session Chair:
Monday, 28/03/2011: 10:30am - 1:00pm
Lecture hall 14
Stephen Andrew Bustin, Michael W Pfaffl
10:30
MIQE: compliance and amendments
Stephen Andrew Bustin1, Jo Vandesompele2
1
Barts and the London School of Medicine, QMUL, United
Kingdom; 2Center for Medical Genetics Ghent (CMGG)
Ghent University Hospital
11:00
Convenient and reliable gene expression profiling
using RealTime ready Focus Panels in combination
with sound data-analysis using qbasePLUS
Ali Rihani1, Tom Van Maerken1, Barbara D'haene2, Nurten
Yigit1, Jo Vandesompele1,2, Rita Rein3; 1Center for Medical
Genetics, Ghent University, Ghent, Belgium; 2Biogazelle,
Ghent, Belgium; 3Roche Diagnostics, Germany
"Stay in touch while on the bench" MIQE qPCR
Afif Abdel Nour1, Michael W. Pfaffl2
1
LaSalle Beauvais, France; 2Physiology Weihenstephan,
TUM, Germany
11:30
12:00
12:30
15:30 – 16:00
16:00
Single Molecule RNA FISH: novel, simple, and
accurate quantitative applications for gene expression
analysis
Arjun Raj
University of Pennsylvania, United States of America
16:30
Relationship between cell attachment and bcr-abl cell
expression heterogeneity within CML cell lines.
Philip Day, Ehsan Karimiani
University of Manchester, United Kingdom
17:00
Digital Competitive Allele-Specific TaqMan-Based
Reverse Transcription-qPCR (castRT-qPCR) for Direct
Detection and Enumeration of Circulating Tumor Cells
David Xingfei Deng, Yun Bao, Scott Sproul, Yu Wang,
Fawn Wang, David Merrill, Pius Brzoska, Caifu Chen
Life Technologies, United States of America
17:20
Real time PCR on slide: a new tool for quantification
of minute amounts of DNA
Jean-Christophe Avarre1, Angelique le Bras2, Régis
Melizzi2, Maxime Rattier2, Gordana Cerovic2, Claude
Weisbuch2, Marianna Alunni3, Martin Kantlehner3, Petra
Hartmann3, Reinhard Bittner3, Wolfgang Mann3
1
Institut de Recherche pour le Développement,
Montpellier, France; 2Genewave SAS, Paris, France;
3
Beckman Coulter Biomedical GmbH, Munich, Germany
17:40
Towards a Comprehensive Single Cell Expression
Profiling
Herbert Auer
IRB Barcelona, Spain
qPCR Gene Expression Assays and an Exogenous
RNA control for indication of reaction inhibition in the
RT-qPCR workflow
Jaakko Kurkela
Thermo Fisher, Finland
Quality control and management approaches for
Molecular Diagnostics techniques used during
diagnosis
and
monitoring
of
hematologic
malignancies
Barbara Zehentner
HematoLogics Inc, United States of America
Coffee break & Networking
18:00 – 21:00
Evening Poster Session
Tuesday 29th March 2011
14:30
Qualitative and Quantitative Analysis of RNA by
SOLiD Next Generation Sequencing
Thomas Rygus
Life Technologies GmbH, Germany;
High throughput & digital PCR
15:00
Analysis of circulating nucleic acids (CNA) using NGS
technologies
Bertram Brenig1, Julia Beck2, Ekkehard Schütz2, Howard B
Urnovitz2
1
University of Goettingen, Germany; 2Chronix Biomedical
GmbH, Germany
15:30
Improved library quantification for High Throughput
Sequencing at the Wellcome Trust of Human Genetics
Oxford
Paolo Piazza
Wellcome Trust Centre for Human Genetics, United
Kingdom
Time:
Location:
Session Chair:
Tuesday, 29/03/2011: 8:30am - 12:00pm
Lecture hall 14
Frank McCaughan
Kevin L Knudtson
8:30
Using Nanoscale PCR and NGS Technologies to
Detect Rare Sequence Variants in a Core Setting
Kevin L Knudtson
University of Iowa, United States of America
9:00
Customized, function tested RealTime ready assays
for gene expression analysis in biomarker research
and early drug development
Sabine Lohmann1, Andrea Herold1, Bergauer Tobias3,
Belousov Anton2, Betzl Gisela1, Dietrich Manuel1, Hessel
Harald5, Poignee-Heger Manuela1, Geho David4, Weisser
Martin2
1
Roche Applied Science, Penzberg, Germany; 2Roche
Pharma, Penzberg, Germany; 3Roche Pharma, Basel,
Switzerland; 4Roche Pharma, Nutley, USA; 5LudwigMaximillian-University, Institue of Pathology, Munich,
Germany
9:30
Digital PCR - getting the most out of difficult clinical
samples
Frank McCaughan
Medical Research Council, United Kingdom
10:00 – 10:30
16:00 – 16:30
16:30
Application of the Next Generation Sequencing
Technologies at JCVI
Yu-Hui Rogers
J. Craig Venter Institute, United States of America
17:00
The
use
of
qRT-PCR
and
high-throughput
transcriptomics for biomarker development
Michael W Pfaffl, Irmgard Riedmaier, Heinrich HD Meyer
Physiology Weihenstephan, TUM, 85354 Freising,
Germany
17:30
Deep sequencing as diagnostic tool for highly
pathogenic viruses
Aleksandar Radonic, Andreas Kurth, Wojtek Dabbrowski,
Andreas Nitsche
Robert Koch Institute, Germany
18:00
High Resolution Transcription Profiling using NextGeneration-Sequencing identifies Diagnostic Markers
for Malignancy
Ralf Horres1,3, Björn Rotter1, Peter Winter1, Michalski
Christoph2, Friess Helmut2, Jüngling Ruth1,3, Günter Kahl3,
Nicolas Krezdorn1, Klaus Hoffmeier1, Jörg Kleeff2
1
GenXPro GmbH, Germany; 2Chirurgische Klinik und
Poliklinik, Klinikum rechts der Isar, TU München; 3Goethe
Universität
Frankfurt,
Institut
für
Molekulare
Biowissenschaften
10:30
Digital PCR or Finding The Needle In A Haystack
Elena Grigorenko
Life Technologies, Inc, United States of America
11:00
Digital Analysis- a bridge to the Clinic
Jim White
NanoString Technologies, United Kingdom
11:30
High-throughput real-time PCR approach using the
LightCycler®1536 instrument for genotyping and high
discrimination of E. coli pathogroups responsible for
gastrointestinal diseases
Sabine Delannoy1, Lothar Beutin2, Cédric Woudstra1,
Patrick Fach1
1
French Agency for Food, Environmental and
Occupational Health & Safety (ANSES), France; 2NRL for
E. coli, Federal Institute for Risk Assessment, Germany
19:00 – 24:00
Location: Lindenkeller Pasta & More, Freising
International Buffet and Asian Buffet
12:00 – 14:00
12:00 – 14:00
Music and Dancing
NGS: Next Generation Sequencing
Time:
Location:
Session Chair:
14:00
Tuesday, 29/03/2011: 2:00pm - 6:00pm
Lecture hall 14
Yuk Ming Dennis Lo, Yu-Hui Rogers
Noninvasive prenatal diagnosis using fetal DNA in
maternal plasma: from digital PCR to next-generation
sequencing
Yuk Ming Dennis Lo
The Chinese University of Hong Kong, China
New qPCR Applications and Method Optimisation
RNAi - microRNA - siRNA Applications
Time:
Location:
Session Chair:
Tuesday, 29/03/2011: 8:30am - 12:00pm
Lecture hall 15
Jo Vandesompele, Jan Hellemans
8:30
Development of an ultra-high throughput long noncoding RNA qPCR screening system
Jan Hellemans1, Pieter Mestdagh2, Steve Lefever2,
Barbara D'haene1, Filip Pattyn2, Frank Speleman2, Jo
Vandesompele1
1
Biogazelle, Ghent, Belgium; 2Ghent University, Belgium
9:00
Towards blood-based cancer screening using LNA™enhanced microRNA qPCR
Ditte Andreasen, Niels Tolstrup, Jacob Ulrik Fogh, Søren
Jensby Nielsen, Kim Bundvig Barken, Adam Baker, Peter
Mouritzen
Exiqon, Denmark
9:30
MicroRNA Target Validation with MISSION® 3'UTR Lenti
GoClone™ and Human MicroRNA Mimics
Nikos Hontzeas
Sigma Aldrich, United States of America
10:00 – 10:30
Time:
Location:
Session Chair:
14:00
Quantitative Immuno-PCR based on Imperacer:
Technology, Platform & Analytical Applications
Jan Detmers, Mark Spengler, Michael Adler
Chimera Biotec GmbH, Germany
14:30
Quantitative Assessment of Chromatin Structure:
Analysis of Epigenetically Regulated Gene Promoters
Francisco Bizouarn
Bio-Rad Laboratories Inc, United States of America
15:00
Quantification of specific mutation with HRM for
regions of interest
Robert Loewe
GeneWake GmbH, Germany
15:30
Quantitation of Rare Undifferentiated hESCs
Methylation-Specific CastPCR (MeS castPCR)
Jason Gioia, David Deng, Jeff Rosner, Caifu Chen
11:00
11:30
Impact of different normalization strategies on miRNA
profiling experiments
Swanhild Meyer1, Sebastian Kaiser2, Carola Wagner3,
Christian Thirion4, Michael Pfaffl1
1
Physiology Weihenstephan, Technische Universität
München, Germany; 2Department of Statistics, LudwigMaximilians-Universität München, Germany; 3IMGM
Laboratories GmbH, Martinsried, Germany; 4FriedrichBaur-Institute and Department of Neurology, LudwigMaximilians-Universität München, Germany
RNAi and gene expression profiling as a tool in cancer
research
Michelle Plusquin1, An-Sofie Stevens1, Katrien De
Mulder2, Frank Vanbelleghem1, Peter Ladurner2, Ann
Cuypers1, Tom Artois1, Karen Smeets1
1
Hasselt University, Belgium; 2University of Innsbruck
Absolute and relative quantification of placental
specific microRNAs in maternal circulation in
placental insufficiency related complications
Ilona Hromadnikova1, Katerina Kotlabova1, Jindrich
Doucha2, Klara Dlouha3
1
Third Faculty of Medicine, Charles University Prague,
Czech Republic; 2Clinic of Obstetrics and Gynecology,
University Hospital Motol, Prague, Czech Republic;
3
Institute for the Care of the Mother and Child, Prague
12:00 – 14:00
12:00 – 14:00
qbasePLUS: data analysis lunch seminar
16:30
PrimeTime® Pre-designed qPCR Assays using ZEN™
Internal Dark Quencher Chemistry
Scott D. Rose
Integrated DNA Technologies, United States of America;
17:00
PrimerXL: high-throughput assay design for qPCR
and amplicon based NGS
Filip Pattyn, Steve Lefever, Frank Speleman, Jo
Vandesompele
Ghent University, Belgium
17:30
Hot Start dNTPs – Novel Chemistries for Use in
Advanced PCR Applications
Natasha Paul
TriLink BioTechnologies, Inc., United States of America
17:45
PCR-Luminex Based Detection of 28 Enteropathogens
Mami Taniuchi1, Jie Liu1, Shihab Sobuz2, William Petri1,
Rashidul Haque2, Eric Houpt1
1
University of Virginia, United States of America;
2
International Centre for Diarrhoeal Diseases and
Research, Bangladesh
18:00
Simple analytical and experimental procedure for
selection of reference genes for qPCR normalization
data
Rodrigo Manjarin1, Nathalie Trottier1, Patty Weber2, James
Liesman1, Nathanael Taylor1, Juan Pedro Steibel1,3
1
Department of Animal Science, Michigan State University,
United States of America; 2Department of Large Animal
Clinical Sciences, Michigan State University, United States
of America; 3Department of Fisheries and Wildlife,
Michigan State University, USA
19:00 – 24:00
Location: Lindenkeller Pasta & More, Freising
Time:
Location:
Session Chair:
13:00
by
16:00 – 16:30
10:30
Tuesday, 29/03/2011: 2:00pm - 6:00pm
Lecture hall 15
Heinrich HD Meyer, Robert Loewe
Tuesday, 29/03/2011: 1:00pm - 2:00pm
Lecture hall 15
Barbara D'haene, Jan Hellemans
qbasePLUS 2.0: the next generation of real-time PCR
data-analysis software
Barbara D'haene, Jo Vandesompele, Jan Hellemans
Biogazelle, Belgium
International Buffet and Asian Buffet
Music and Dancing
Wednesday 30th March 2011
14:00
FRET primer for single nucleotide polymorphism
(SNP) genotyping
Choy Len Fong1, Fiona Ng1, Kanaga Sabapathy2, Kim
Halpin1
1
Life Technologies, Singapore; 2Laboratory of Molecular
Carcinogenesis, National Cancer Centre, Singapore;
14:15
qPCR for the Detection and Quantification of AdenoAssociated Virus Serotype 2 (AAV2) ITR-Sequences
Christine Aurnhammer, Maren Haase, Anja Ehrhard,
Martin Häusel, Ingrid Huber, Nadine Muether, Hans
Nitschko, Ulrich Busch, Andreas Sing, Armin Baiker
Bavarian Health and Food Safety Authority (LGL),
Germany
14:30
Cost-efficient detection of Mycobacterium avium
subsp. paratuberculosis in faeces by quantitative real
time PCR and development of a predictive model for
fighting the disease
Petr Kralik1, Iva Slana1, Alena Kralova1, Vladimir Babak1,
Robert H. Whitlock2, Ivo Pavlik1
1
Veterinary Research Institute, Czech Republic;
2
University of Pennsylvania, School of Veterinary
Medicine, PA, USA
Pre-PCR: Pre-analytical Steps
Time:
Location:
Session Chair:
Wednesday, 30/03/2011: 9:00am - 12:00pm
Lecture hall 14
Jo Vandesompele
Karl H. Hasenstein
9:00
Measurable impact of RNA quality on gene expression
results from quantitative PCR
Joëlle Vermeulen1, Katleen De Preter1, Steve Lefever1,
Justine Nuytens1, Fanny De Vloed1, Stefaan Derveaux1,
Jan Hellemans2, Frank Speleman1, Jo Vandesompele2
1
2
Ghent
University,
Ghent,
Belgium;
Biogazelle,
Zwijnaarde, Belgium & Ghent University, Ghent, Belgium
9:30
Isolation and Analysis of Circulating Nucleic Acids:
Technical Advances
Martin Horlitz
QIAGEN GmbH, Germany;
10:00
Solid Phase Gene Extraction – Sampling of mRNA
from living systems
Karl H. Hasenstein, Min Chen
Univ. Louisiana Lafayette, United States of America
10:00 – 10:30
Impact of basepair mismatch in primer annealing on
qPCR assay performance
Steve Lefever, Pattyn Filip, Speleman Frank, Hellemans
Jan, Vandesompele Jo
Center for Medical Genetics Ghent, Ghent University,
Belgium
11:00
Gene Expression profiles from FFPE samples with
improved RNA decrosslinking technology A case
study: Molecular profiling of breast cancer from
formalin-fixed, archival material
Guido Krupp1, Susanne Quabius2, Rolf Jaggi3
1
AmpTec GmbH, Hamburg, Germany; 2Institute for
Immunology, University Klinikum UK-SH, Kiel, Germany;
3
Dept. Clinical Research, University of Bern, Bern,
Switzerland
Development of a novel duplex real-time PCR assay
for plasma DNA quantification and its comparison to
other methods
Shiyang Pan, Dan Chen, Peijun Huang, Bing Gu, Fang
Wang, Jian Xu, Chun Zhao, Yongqian Shu, Di Yang
The first affiliated Hospital with Nanjing Medical University,
China
15:30
Autocrine and paracrine role of leptin and
thrombopietin in controlling ovarian function in cow
Mihir Sarkar1,2, S Schilffarth1, D Schams1, HHD Meyer1, M
W Pfaffl1, S Ulbrich1, B Berisha1,3
1
Technical University Munich, Germany; 2Physiology &
Climatology, Indian Veterinary Research Institute,
Izatnagar, Bareilly, Uttar Pradesh, India; 3Faculty of
Agriculture and Veterinary, University of Prishtina,
Prishtine, Kosovo
15:45
ULTRA-RAPID REAL-TIME PCR for the detection of
viral diseases in honeybee
ByoungSu Yoon, MiSun Yoo, JiNa No, Van Phu Nguyen
Kyonggi University, South Korea
16:00
Development of quantitative triplex real time PCR for
the simultaneous detection of Mycobacterium avium
subsp. avium and M. a. subsp. hominissuis
Iva Slana, Maria Kaevska, Petr Kralik, Alice Horvathova,
Ivo Pavlik
Veterinary Research Institute, Czech Republic
16:15
Rapid KRAS, EGFR, BRAF and PIK3CA Mutation
Analysis of Fine Needle Aspirates using allele-specific
qPCR
Ronald van Eijk, Jappe Licht, Hans Morreau, Tom van
Wezel
Leiden University Medical Center, The Netherlands
DEPArray™: a novel platform to identify, isolate and
collect very rare cells
Pim van der Aar1, Manuela Banzi2
1
Dync B.V., Netherlands, The; 2Silicon Biosystems SpA;
12:00 – 14:00
12:00 – 14:00
15:15
10:30
11:30
14:45 – 15:15
New qPCR Applications in Molecular Diagnostics
Lecture hall HS 14
Time:
Location:
Session Chair:
13:45
Wednesday, 30/03/2011: 13:45pm - 4:30pm
Lecture hall 14
Andreas Nitsche
Heinrich HD Meyer
Detection and classification of microorganisms by
combination of qPCR and pyrosequencing
Kati Schroeder, Andreas Nitsche
Robert Koch-Institut, Germany
16:30
GENEX: data analysis lunch seminar
Data Analysis: qPCR BioStatistics &
BioInformatics
Time:
Location:
Session Chair:
Wednesday, 30/03/2011: 9:00am - 12:00pm
Lecture hall 15
Michael W Pfaffl, Ales Tichopad
9:00
Interpretation requires context - making sense out of
gene lists and networks
Philip Zimmermann
ETH Zürich, Switzerland
9:30
Stratified error in the qPCR assays from the statistical
point of view
Ales Tichopad
Academy of Science of Czech Republic, Czech Republic
10:00
Primer3: Improvements for the design of qPCR
primers
Andreas Untergasser1, Ioana Cutcutache2, Steve Rozen2
1
University of Heidelberg, Germany; 2Duke-NUS Graduate
Medical School, Singapore
10:30 – 11:00
11:00
11:20
Time:
Location:
Session Chair:
13:00
Presentation of GenEx 5.3; the most user friendly and
powerful qPCR experimental design, data analysis
and data mining software yet released!
Mikael Kubista
MIQE: state of the art & open discussion
Time:
Location:
Session Chair:
Wednesday, 30/03/2011: 2:00pm - 3:00pm
Lecture hall 15
Stephen Andrew Bustin
Jo Vandesompele
15:00 – 15:30
Is normalisation of raw data necessary? Truly
Automated Analysis of qPCR Data Using the
AzurePCR Method
David Kennard, Ze'ev Russak
Azure PCR Limited, United Kingdom
Amplification efficiency as a function of primer and
cDNA concentration
Jan M Ruijter1, Quinn D Gunst1, Peter Lorenz2, Maurice JB
vandenHoff1
1
Academic Medical Center, Amsterdam, Netherlands, The;
2
University of Rostock, Rostock, Germany
11:40
Evaluation and applicability of advanced/exotic qPCR
quantification strategies and their implementation
Andrej-Nikolai Spiess
University Hospital Hamburg-Eppendorf, Germany
12:00
High-resolution melting data error evaluation between
runs.
Maksim Bratchikov, Mykolas Mauricas
Centre for Innovative Medicine, State Research Institute,
Vilnius, Lithuania
12:00 – 14:00
12:00 – 14:00
Wednesday, 30/03/2011: 1:00pm - 2:00pm
Lecture hall 15
Mikael Kubista
Amin Forootan
RDML: state of the art & open discussion
Time:
Location:
Session Chair:
Wednesday, 30/03/2011: 3:30pm – 4:30pm
Lecture hall 15
Jan Hellemans
Andreas Untergasser
Lecture hall HS 14
16:30
Thursday 31st March & Friday 1st April 2011
qPCR Application Workshops
The workshops are aimed at giving participants a deep and objective understanding of real-time quantitative PCR, biostatistics, expression
profiling, and its applications. The courses are intended for academic or industrial persons considering working with qPCR or scientists currently
working with qPCR seeking a deeper understanding.
The qPCR courses cover all aspects in qPCR and are held during 2-days. Each course is approximately 50% hands-on and is limited to 20-25
participants (biostatistics 40-50 participants), resulting in very interactive teaching and everybody given the opportunity to try the
instrumentation. After the course participants will be able to plan and perform qPCR experiments themselves, as well as interpret and analyze
data. Detailed course material and full catering (lunch, coffee, soft drinks and snacks) are included in the course fee.
All workshops start on Thursday and Friday at 9 am until 5 pm. All three workshops are hosted by the TATAA Biocenter Sweden, and bioEPS
GmbH (www.tataa.com www.bioeps.com). The qPCR workshop laboratories and seminar rooms are close to the central lecture hall.
Workshop topics:


Basic Module qPCR Application Workshop (2-days)
qPCR Biostatistics & Expression Profiling Workshop (2-days)
Practical room – P1
Computer seminar room – PU

MIQE guidelines (1 day) & Practical primer design (1-day)
Seminar room - S1
Basic Module qPCR Application Workshop (2-days)
Practical room – P1
The introductory course consists of a theoretical part and a practical part where participants get to do QPCR experiments by themselves under
experienced supervision. The course contains:
Day 1 - hands on workshop:
• Basic PCR theory
• The theory of real-time PCR
• Applications and possibilities of qPCR. Comparison of qPCR with regular PCR.
• Review of currently available detection technologies (SYBR Green I, hydrolysis probes, Molecular Beacons...etc)
• Different instrument platforms and their typical uses
• Primer design
• The problem of primer-dimer formation and how to minimize them
• Probe design
• Experimental design and optimization
• Basic data handling and analysis
Day 2 - hands on workshop:
• Introduction to quantification principles
• Quantification strategies, uses and limitations
• Calculations using different relative quantification methods
• Strategies for normalization of qPCR data
• In situ calibration for compensation of inhibition in samples
• Absolute quantification
qPCR data analysis - Biostatistics & Expression Profiling (2-days)
Computer seminar room – PU
This course will teach you how appropriate statistics should be selected and applied correctly to get the most out of your qPCR data. The course
mixes theoretical lectures with computer based exercises. Please bring your own Laptop to the course!
Day 1- Statistical analysis of real-time PCR data
• Basic principles of statistics
• Advanced principles of statistics
• Statistical tests
• Ability to detect a difference
Day 2- Gene expression profiling with real-time PCR
• Multiplate measurements
• Standard curves and absolute quantification
• Experimental design, selecting reference genes
• Relative quantification, comparison of groups
• Expression profiling
MIQE guidelines (1 day) & Practical primer design (1-day)
Seminar room - S1
Day 1 - The MIQE guidelines – How to improve your qPCR quality
This is a theoretical course that will take you through the MIQE guidelines. The MIQE guidelines (Minimum Information for Publication of
Quantitative Real-Time PCR Experiments) have been developed to ensure the integrity of the scientific literature, promote consistency between
laboratories and increase experimental transparency. The course will explain the different steps that are necessary to fulfill to ensure good
qPCR quality and show you how it can be done. The course includes:
•
•
•
•
•
•
•
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Introduction to the MIQE guidelines
Sample preparation
Nucleic acid extraction and quality control
Reverse Transcription
Primer and probe design
Optimization and validation of qPCR
Normalization
qPCR data analysis
Day 2 - Practical primer design
This is an extensive primer design course that will teach you how to design your own assays. It will teach you the theory of primer and probe
design as well as how to optimize your reactions. The course will also let you do design of your own primers using primer design softwares
under experienced supervision.
qPCR Workshop Sponsors:
35 companies participate at the industrial exhibition held during the qPCR Symposium March 28th – 30th in the foyer of the central lecture hall
complex (green frame) and in two side rooms S1 and S2 (blue frame).
Booth Company
Booth
Company
1&2
3
4
5
6
7
8
9
10
11
12
13
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15
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17
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Life Technologies
Fujifilm
MMI
Eurogentec
Lonza
Sigma Life Science
Amplifa
Fluidigm
Biolegio
TATAA & MultiD
Wafergen
ArcticZymes
Biogazelle
Analytik Jena & Biometra
Primerdesign
Affymetrix
Illumina
33
34
Registration & Catering
32
S2
31
26
29
30
25
28
S1
27
Kitchen
24
Agilent Technologies
Genewave
Bio-Rad
Qiagen
Nanostring
Roche Applied Science
Thermo Fisher Scientific
Biosearch Technologies
Exiqon
Eurofins MWG Operon
TaKaRa Bio Europe
Metabion
Bioke
TIB Molbiol
Integrated DNA Technologies
Bioline
4titude
Rest
Rooms
Rest
Rooms
Main Exhibition Area
Booth - Foyer
in Foyer
Booth - S1 & S2
Lecture hall
HS 15
2
Le
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ct u
HS re h
1 4 a ll
5
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Abstracts - Oral presentations
th
Monday 28 March 2011
MIQE and QC strategies in qPCR
Time:
Location:
Session Chair:
Monday, 28/03/2011: 10:30am - 1:00pm
Lecture hall 14
Stephen Andrew Bustin, Michael W Pfaffl
MIQE: compliance and amendments
Stephen Andrew Bustin1, Jo Vandesompele2
1
Barts and the London School of Medicine, QMUL, United
Kingdom; 2Center for Medical Genetics Ghent (CMGG) Ghent
University Hospital
The publication of the minimum information for the publication of
real-time quantitative PCR experiments (MIQE) guidelines has
become a defining event in the maturing of qPCR technology. The
response from instrument and reagent manufacturers has been
universally positive; there has been extensive publicity in print,
online and at scientific meetings and many scientific journals are
beginning to take note. We report the results of the first extensive
survey carried out to determine transparency and quality of recent
qPCR publications. It has also become apparent that an important
obstacle to the universal acceptance of the MIQE guidelines has
been the stipulation for primer sequence disclosure. Many
commercial qPCR assays do not provide this information; usually
there are also no details provided on empirical validation of each
individual assay. The increasing use of commercial qPCR assays
is creating problems, as it results in publications that cannot
satisfy current MIQE requirements. Consequently, we propose an
amendment of the original guidelines to help users of commercial
assays publish transparent protocols that fulfil the MIQE criteria.
Convenient and reliable gene expression profiling
using RealTime ready Focus Panels in combination
with sound data-analysis using qbasePLUS
Ali Rihani1, Tom Van Maerken1, Barbara D'haene2, Nurten
Yigit1, Jo Vandesompele1,2, Rita Rein3
1
Center for Medical Genetics, Ghent University, Ghent, Belgium;
2
Biogazelle, Ghent, Belgium; 3Roche Diagnostics, Germany
Roche’s RealTime ready Focus Panels are ready-to-use, prevalidated qPCR assays targeting selected genes from specific
pathways or functional groups. We aimed to evaluate the utility of
the technology for convenient and reliable gene expression
analysis in accordance to the MIQE guidelines. A pilot experiment
was set up using the human apoptosis panel that enables
expression profiling of 372 genes. Two previously established
chemosensitive and chemoresistant neuroblastoma cell lines were
analyzed before and after treatment with the MDM2 inhibitor
nutlin-3. Thorough post-qPCR quality control and data-processing
was performed using Biogazelle’s qbasePLUS software. We were
able to demonstrate (1) the expression stability of a selected
subset of the included reference genes, (2) the utility of the
integrated positive and negative RT controls and RNA integrity
assessment, (4) the ease and power of data analysis using
qbasePLUS, and (3) the power of the strategy to obtain biologically
relevant results when scrutinizing an entire pathway. Overall, the
study demonstrates that the RealTime ready Focus Panels in
combination with the state-of-the-art real-time PCR data-analysis
software qbasePLUS enable easy, fast, and reliable gene
expression quantification.
"Stay in touch while on the bench" MIQE qPCR
Afif Abdel Nour1, Michael W. Pfaffl2
1
LaSalle Beauvais, France; 2Physiology Weihenstephan, TUM,
Germany
Who does not know iPhone and iPad devises? More and more
people have an Apple mobile device such as iPhone, iPad or iPod
touch. From now on, those mobile devices can be used to
increase quality of qPCR experimant or publication, by providing a
'MIQE APP'. This new application could be used by scientists to
check whether their experiment or the used literature fulfills the
MIQE requirement or not. The 'MIQE Guidelines' checklist
provides 85 parameters that qPCR studies should be required or
recommended to meet before being considered for publication.
This checklist is based on the original published MIQE checklist
and we hope it will increase future publication quality and
reliability. But there are much more wider seen goals of the 'MIQE
guidelines', all in all the goals might be summarized; to increase
reliability of results to help to insure the integrity of scientific work,
with major focus on biological relevance. The MIQE applet will
provide an interactive and portable follow up of these parameters
for any qPCR research application. The applet will be free for
download and upon installed connected to our MIQE databank,
collecting all the information. The 'MIQE guidelines' (Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments) were published by a group of qPCR experts in 2009
(Bustin et al, Clinical Chemistry 2009).
qPCR Gene Expression Assays and an Exogenous
RNA control for indication of reaction inhibition in the
RT-qPCR workflow
Jaakko Kurkela
Thermo Fisher, Finland
Reverse transcriptase quantitative PCR (RT-qPCR) is a routine
laboratory technique for the measurement of RNA that is both
sensitive and specific with appropriate assay design. Thermo
Scientific Solaris qPCR Gene Expression Assays are probe-based
assays that are ideal for routine molecular applications; they
combine minor groove binder technology (MGB) with a rigorous
algorithm that permits detection of all known splice variants of a
gene target, while distinguishing among closely related family
members, on a genome-wide scale. Inhibitors of RT-qPCR may
be present in experiments and not easily recognized. The primary
challenge is that methods such as spectrophotometric techniques
do not always detect RT-qPCR inhibitors. The Thermo Scientific
Solaris RNA Spike Control Kit was designed as an exogenous
control to identify the presence of reaction inhibition including
those commonly carried through processing steps to isolate RNA
such as EDTA, phenol, heparin and EtOH. Here we describe
applications for this exogenous control to identify the presence of
reaction inhibition and illustrate the impact inhibition can have on
results and data interpretation. Experimental results demonstrate
RNA Spike Control provides accurate identification of RT-qPCR
inhibition and the ability to predict when inhibition has been
removed, for example, by dilution of the RNA sample into the
reverse transcription reaction. The RNA Spike Control identified
RT-qPCR inhibition in a gene expression assay, explaining the
variable results obtained. Furthermore, the use of RNA Spike
Control Kit was compared to current inhibition identification
protocols and was found to be an easy and succinct workflow,
offering a clear advantage of this technology.
Quality control and management approaches for
Molecular Diagnostics techniques used during
diagnosis and monitoring of hematologic
malignancies
Barbara Zehentner
HematoLogics Inc, United States of America
This presentation will provide an introduction to current
applications of molecular analysis tools in the clinical field
of hematologic malignancies. Several different assay platforms will
be covered, from B- and T-cell clonality assessment by gene
rearrangement PCR, point mutation analysis for myeloid
neoplasms, Sanger sequence analysis for CLL prognosis and
Gleevec resistance assessment, in addition to the application of
real-time PCR for quantitative monitoring purposes and minimal
residual disease detection. Quality control and management
approaches for the clinical reference laboratory will be discussed.
Substantial performance discrepancies between
commercially available kits for rt-qPCR - A systematic
comparative investigator-driven approach
Matthias W Sieber1,2, Peter Recknagel2, Florian Glaser2, Otto
W. Witte1, Michael Bauer2, Ralf A. Claus2, Christiane Frahm1
1
Hans Berger Clinic for Neurology, Jena University Hospital,
Germany; 2Anaesthesiology and Intensive Care Medicine, Jena
University Hospital, Germany
Reverse transcription followed by quantitative PCR (rt-qPCR) has
become the state of the art tool forquantification of nucleic acids.
However, there are still significant problems associated with its
sensitivity, reproducibility, efficiency and the choice of an
appropriate rt-qPCR kit. The purpose of this study is to give
insights into strategies to optimize and validate the performance of
currently available kits for rt-qPCR and to provide up-to-date
information about the benefits, potentials and pitfalls of rt-qPCR
assays. A selection of 9 cDNA synthesis and 12 qPCR kits were
tested using samples obtained from three species (mouse, rat and
human) and three transcripts (Gapdh, Actb and Hmbs) under
highly standardized conditions. Kits with an outstanding
performance were further analysed to identify the dynamic range
for a reliable quantification of mRNA. Reverse transcription
efficiency varied up to 90 fold depending on the choice of reverse
transcriptase, priming strategy and assay volume. The qPCR kit
test revealed variations in mean relative amplification efficiency
ranging from 54% to 171%. We conclude that currently available
kits for rt-qPCR vary considerably. However, with an appropriate
validation strategy and knowledge about capabilities of a particular
kit, sensitivity, efficiency and reliability could be significantly
improved.
single-cell qPCR
Time:
Location:
Session Chair:
Monday, 28/03/2011: 2:00pm - 6:00pm
Lecture hall 14
Mikael Kubista, Anders Ståhlberg
25 years of PCR - from idea to subcellular expression
profiling
Mikael Kubista
PCR was born from a great idea conceived by Kary Mullis and
later refined into qPCR by Russ Higushi. Specificity was added by
Ken Livak. The technique found immediate use in genetic
engineering leading to several breakthrough innovations, and later
PCR became the preferred platform for clinical diagnostics. The
quantitative aspect of qPCR became particularly valuable in
biological and medical research, and is today key technology in
systems biology. Moreover, the extreme sensitivity of qPCR,
allowing the detection of single molecular copies, and the relative
ease of use, which allows qPCR to be integrated in a streamlined
high throughput workflow, has led to the exciting area of single cell
expression profiling. An astonishing heterogeneity in transcript
levels among seemingly like cells has been found, while
correlation between genes’ expression is a fingerprint of the type
of cell. Most recently, qPCR tomography has been developed to
measure intracellular mRNA profiles, revealing gradients of
transcripts within the cell, preparing it for asymmetric cell division.
Anders Stahlberg, Daniel Andersson, Johan Aurelius, Maryam Faiz,
Marcela Pekna, Mikael Kubista, Milos Pekny. Defining cell populations with
single-cell gene expression profiling: correlations and identification of
astrocyte subpopulations. Nucl. Acids Res. 1–12. doi:10.1093/nar/gkq1182
(2010).
Radek Sindelka, Monika Sidova, David Svec, Mikael Kubista. Spatial
expression profiles in the Xenopus laevis oocytes measured with qPCR
tomography. Methods 51:87-91(2010).
M. Bengtsson, A. Ståhlberg, P. Rorsman, and M. Kubista Gene expression
profiling in single cells from the pancreatic islets of Langerhans reveals
lognormal distribution of mRNA levels. Genome Research 15, 1388-1392
(2005).
Research Highlights in Nature Review Genetics 6, 1758 (2005).
Uncovering the Diversity of Individual Cells: Gene
Expression Profiling with the BioMark System
Ken Livak
Fluidigm Corporation, United States of America
Fluctuations in gene expression at the single cell level could be
key for generating developmental signals and for understanding
the progression of tumors. Data needs to be collected from a
statistically significant number of single cells in order to determine
the range of gene expression present in a population of cells.
Furthermore, transcripts need to be quantified for a number of
genes in order to obtain meaningful cell signatures. BioMark™
arrays provide a convenient and cost-effective system for
performing multiple RNA expression assays on multiple single-cell
samples. This system has been used to study single cell gene
expression in embryonic stem cells, hematopoieticstem cells,
cancer stem cells, and early stage embryos.
Identification of unknown cell populations and
correlations using single-cell gene expression
profiling
Anders Ståhlberg
University of Gothenburg, Sweden
Single-cell gene expression levels show substantial variations
among cells in seemingly homogenous populations. Access to
fundamental information about cellular mechanism, such as
correlated gene expression, motivates studies of multiple genes
expressions in individual cells. Astrocytes perform many control
and regulatory functions in the central nervous system. In contrast
to neurons, we have limited knowledge about functional diversity
of astrocytes and its molecular basis. We will show how
subpopulations of cells can be identified at single-cell level using
unsupervised algorithms and that gene correlations can be used
to identify differences in activity of important transcriptional
pathways. We identified two subpopulations of astrocytes with
distinct gene expression profiles. One had an expression profile
very similar to that of neurosphere cells, whereas
the other showed characteristics of activated astrocytes in vivo.
Technical considerations related to reproducible and efficient
sampling, lysis, reverse transcription and real-time PCR will also
be presented. In addition to astrocytes, single-cell data from tumor
cells, beta-cells and embryonic stem cells will be shown.
Single Molecule RNA FISH: novel, simple, and
accurate quantitative applications for gene expression
analysis
Arjun Raj
University of Pennsylvania, United States of America
We have developed a simple, direct method for specifically
detecting individual RNA molecules in situ via fluorescence
microscopy. Our RNA-FISH method relies on using large numbers
of fluorescently labeled oligonucleotides designed to target
particular RNA species. This approach is highly specific and highly
sensitive and provides absolute, direct quantification of RNA
abundance in single cells. Moreover, it is applicable to a broad
range of biological sample types, ranging from microbes to human
tissue section. We will discuss the method itself as well as
address some biological applications in development and cancer.
Relationship between cell attachment and bcr-abl cell
expression heterogeneity within CML cell lines.
Philip Day, Ehsan Karimiani
University of Manchester, United Kingdom
Chronic myelogenous leukemia (CML) is believed to occur as a
consequence of the clonal expansion of leukemic stem cells and
to be maintained by an expanding population of hematopoietic
stem cells that have acquired a BCR-ABL fusion gene. Recent
studies indicate that primitive CML cells are less responsive to
tyrosine kinase inhibitors and are a reservoir for the emergence of
tyrosine kinase resistant subclones. Some studies have
suggested that expression of BCR-ABL may also be required for
altered cell adhesion and CML progression. The bcr-abl protein
modulates cell adhesion and its effects in cell lines like K562
correlate with increased adhesion to fibronectin. It also has been
reported that cell adhesion mediated resistance to apoptosis
induced by BCR-ABL inhibitors, suggests that bcr-abl mediated
cell adhesion may be involved in post-therapy residual disease of
CML. However, some reports imply that a relatively small fraction
(2%–20%) of blasts from patients with acute myeloid leukemia
adhere to the plastic of the cell-culture dish. In this study we test if
elevated BCR-ABL expression could be specifically identified in
individual cells from attached cell populations and not unattached
populations. The study developed a means to utilise flow assisted
cell sorting of cell lines expressing BCR-ABL to derive individual
attached and unattached cell sub-populations. Using a
homogenous extraction procedure, cells individually flow sorted
into microtitre plates were subjected to combinations of abl and
bcr-abl qRT-PCR, and revealed the existence of low and high bcrabl expressing cell line populations. The implications of this study
will be discussed.
Digital Competitive Allele-Specific TaqMan-Based
Reverse Transcription-qPCR (castRT-qPCR) for Direct
Detection and Enumeration of Circulating Tumor Cells
(CTCs)
David Xingfei Deng, Yun Bao, Scott Sproul, Yu Wang, Fawn
Wang, David Merrill, Pius Brzoska, Caifu Chen
Molecular characterization and enumeration of circulating tumor
cells (CTCs) promise to be valuable for cancer cell diagnosis,
survival prognosis, and treatment guidance. However, current
molecular assays require extensive blood sample enrichment
process before analyzing extraordinarily rare CTCs. Here we
reported a new approach for direct CTC molecular detection in
whole blood samples without prior biophysical processing by
combining sample partition and digital assay of a highly specific
castRT-qPCR. CastPCR can detect rare copies of mutant alleles
with a wide dynamic range of more than 6-log orders and < 5-copy
sensitivity. Whole blood samples with spiked-in known mutation
lung cancer cell lines and from lung cancer patients were
partitioned in aliquots of 2.5 μL – 50 μL onto 96- or 384-well
plate(s), such that each well contains either one cancer cell or
none with per well 20,000 – 400,000 normal white blood cells and
10 – 200 million red blood cells. RNAs and/or DNAs were
extracted by magnetic beads and directly or were pre-amplified
prior to mutation detection. Genetic mutations and cell type
specific markers (such as CK19 and/or CEA) for CTC identification
and enumeration were determined by castRT-qPCR and/or
TaqMan Gene expression assays. The sample partition process
resulted in a relative CTC enrichment or digital enrichment of 20 –
400 folds (the relative ratio of CTC to normal cells) in a CTCpositive well. CastPCR clearly identified known mutation and
CK19 in spiked-in samples of about 10 – 30 cells per mL whole
blood, but detected no mutation signals in any sample well without
cell spiked-in. Furthermore, cell type specific markers (CK19) and
known EGFR mutation(s) were identified in the same sample
wells, suggesting identified mutation is specific and from cancer
cells not from normal cells. In five blood samples from lung cancer
patients, EGFR mutation (p.L858R) was detected in all samples.
Approximately 50% of circulating lung tumor cells in a patient with
positive EGFR p.L858R mutation had also positive EGFR
p.T790M mutation, an inducible drug-resistant CTC marker. For
those samples with negative detection of EGFR mutation,
corresponding wild type sequences were detected in all sample
wells, suggesting the normal DNA amplification of those mutationnegative wells. Our data suggest that combination of digital
sample enrichment and castRT-qPCR can be used to directly
enumerate CTCs and detect cancer-related mutations in whole
blood without prior biophysical sample enrichment. The new
approach paves way for noninvasively CTC monitoring and
individualized therapy.
Real time PCR on slide: a new tool for quantification of
minute amounts of DNA
Jean-Christophe Avarre1, Angelique le Bras2, Régis Melizzi2,
Maxime Rattier2, Gordana Cerovic2, Claude Weisbuch2,
Marianna Alunni3, Martin Kantlehner3, Petra Hartmann3,
Reinhard Bittner3, Wolfgang Mann3
1
Institut de Recherche pour le Développement, Montpellier,
France; 2Genewave SAS, Paris, France; 3Beckman Coulter
Biomedical GmbH, Munich, Germany
Detection, identification and quantification of micro-organisms
represents one of the major challenges for our modern society. It
concerns a wide variety of applications including bioprocess
control, food technology, health care, environmental analysis and
of course clinical diagnostics. In this regard, miniaturization of
PCR protocols may offer many advantages including short assay
time, high precision and sensitivity, high-throughput, low reagent
consumption and portability. Though on-chip PCR is likely to
become the 'next-generation PCR', translating microfluidics to
biology labs is still limited by its cost and the expertise it requires.
In this context, a new format system has been developed, which
allows DNA extraction and real-time PCR amplification in 1-µl
"reaction sites" on a slide. The system, called SicLive, enables to
process 48 samples in parallel and offers the possibility to perform
quantitative PCR amplifications within 2 hours, including DNA
extraction. Results, obtained on different model systems, showed
that reproducibility and dynamic range were comparable to those
obtained in 20 µl with conventional real-time PCR. In terms of
sensitivity, SicLive was able to amplify DNA from 1-5 copies of
target as well as from single cells. By combining DNA extraction
and amplification in a single reaction site, SicLive is particularly
well adapted for quantifying precious samples containing very low
amounts of genetic material, such as clinical or environmental
samples. Finally, for single-cell applications, this slide format
ensures an easy visual control, with standard microscopy, of both
quality and quantity of the templates loaded.
Towards a Comprehensive Single Cell Expression
Profiling
Herbert Auer
IRB Barcelona, Spain
Expression profiling, the measurement of all transcripts of a cell, is
currently the most comprehensive method to describe its
physiological state. Given that accurate profiling methods currently
available require RNA amounts found in thousands to millions of
cells, many fields of biology working with specialized cell types
cannot use these techniques because available cell numbers are
limited. Currently available alternative methods for expression
profiling from picograms of RNA or from very small cell
populations lack a broad validation of results to provide accurate
information about the measured transcripts. Except for a few
highly experimental methods like Single-Molecule-Sequencing,
every attempted measurement of more than a few different
transcripts needs a pre-amplification of cDNA. Here we present
Pico Amplification, a novel workflow of RNA isolation and cDNA
amplification for cell populations as small as a few or even a single
cell. Micrograms of cDNA are generated, suitable for analysis by
qPCR, microarrays or sequencing. Expression profiling of RNAs
diluted to picograms showed that Pico Amplification virtually did
not alter measurements of differential expression. MAQC samples
A and B (Universal Reference RNA and Human Brain RNA) were
utilized to provide transcriptome wide evaluation using microarrays
and for comparison to qPCR measurements for over 800
transcripts. RNA isolated from 10 cells provided after Pico
Amplification virtually identical information about the entire
population as standard protocols do from thousands or millions of
cells. This held true across the entire transcriptome. RNA isolated
from individual cells provided insight into the heterogeneity of
seemingly homogenous cell populations. Hundreds of transcripts
were found to be differentially expressed between individual cells.
Pico Amplification provides micrograms of cDNA from very small
cell populations or even individual cells, highly representative of
the original transcriptome. This allows the application of virtually
all standard methods of expression profiling to interrogate the
transcriptional status of an individual cell.
Tuesday 29th March 2011
High throughput & digital PCR
Time:
Location:
Session Chair:
Tuesday, 29/03/2011: 8:30am - 12:00pm
Lecture hall 14
Frank McCaughan
Kevin L Knudtson
mouse model. Xenograft-derived fresh frozen (FF) tissue samples
were used to test for selected biomarkers. Next, we moved to
early clinical development using the first set of clinical samples,
formalin-fixed paraffin-embedded (FFPE) tissue derived from
various tumor entities. Tumor-derived FFPE tissue is the most
relevant sample material for the development of therapeutic
compounds in clinical trials or in biomarker research. RNA
extraction from FFPE tissue (e.g., 10 µm sections) using the High
Pure RNA Paraffin Kit, followed by qRT-PCR analysis, is a well
established technique; here, we combined this workflow with
function tested RTR assays.
For life science research only. Not for use in diagnostic procedures.
Using Nanoscale PCR and NGS Technologies to
Detect Rare Sequence Variants in a Core Setting
Kevin L Knudtson
University of Iowa, United States of America
The University of Iowa DNA Facility has been providing sequence
variant detection services using nanoscale PCR and NGS
technologies for nearly three years. Detection of sequence
variants that occur in a small percentage of the population has met
with both technical and analytical challenges in avoiding or
minimizing false calls. False positive calls can be very expensive
to verify. False negative calls simply cannot happen as they result
in a missed diagnosis. Using the Applied Biosystems OpenArray
and Fluidigm Dynamic arrays, the ability of each platform to give
the correct genotype for 32 assays over 575 individuals was
evaluated. Both platforms were similar in their false call rate and
the correct call rates improved with by conducting a specific target
enhancement of the samples prior to the genotyping assay.
Variant detection was also evaluated using NGS technologies.
Amplicons from 300 individuals interrogating over 210 target
regions from each individual were mixed together and run over the
Roche GS FLX and Applied Biosystems SOLiD genome
sequencers. False positive rates were initially high, but decreased
as the specificity of the primers was improved. Workflows and/or
experimental design enhancements to reduce false genotyping
call rates will be discussed.
Customized, function tested RealTime ready assays
for gene expression analysis in biomarker research
and early drug development
Sabine Lohmann1, Andrea Herold1, Bergauer Tobias3,
Belousov Anton2, Betzl Gisela1, Dietrich Manuel1, Hessel
Harald5, Poignee-Heger Manuela1, Geho David4, Weisser
Martin2
1
Roche Applied Science, Penzberg, Germany; 2Roche Pharma,
Penzberg, Germany; 3Roche Pharma, Basel, Switzerland; 4Roche
Pharma, Nutley, USA; 5Ludwig-Maximillian-University, Institue of
Pathology, Munich, Germany
Personalized Health care (PHC) uses gene-based information to
understand requirements for health maintenance, disease
prevention, and therapy, all tailored to an individual’s genetic
uniqueness. Biomarkers are therefore a central element
throughout a drug’s lifecycle, from target identification to drug
application. In biomarker research and early drug development,
the investigation of mRNA expression profiles is of great interest.
Roche RealTime ready custom panels offer a broad variety of
intron-spanning RT-qPCR assays covering the major signal
transduction pathways, including those relevant for oncology
research. RealTime ready custom panels are comprised of readyto-use LightCycler® 480 Multiwell Plates containing pre-plated
qPCR assays for human, mouse, and rat targets that have been
selected by the user. We have established various workflows
applicable for gene expression analysis in biomarker research as
well as in pre-clinical and early clinical studies. Pre-clinical drug
development was started by profiling gene expression using a
multi-parameter panel (93 target and 3 housekeeping genes)
covering the pathway of interest in a broad screening approach.
The goal was to generate a first hypothesis for predictive and
pharmacodynamic markers, as well as to provide a workflow with
the highest convenience and throughput for this screening
approach. This first step was performed in vitro with tumor cell
lines treated with the compound of interest. A workflow protocol
was developed, starting with an automated RNA extraction on the
MagNA Pure LC Instrument in combination with pre-plated
RealTime ready customized assays in a 384-well format. The
relative gene expression had been analysed. After selecting a set
of parameters, this first hypothesis was verified using an in vivo
LIGHTCYCLER, MAGNA PURE, HIGH PURE, and REALTIME ready are
trademarks of Roche.
Digital PCR - getting the most out of difficult clinical
samples
Frank McCaughan
Medical Research Council, United Kingdom
We use digital PCR to interrogate clinical biopsy samples both for
basic research and translational medicine purposes. For this we
use a technique called Molecular Copy-number Counting which is
essentially a multiplex hemi-nested single molecule digital PCR
protocol. The advantages of MCC are that we can use it to extract
multilocus information from tiny quantities of starting DNA
extracted from formalin-fixed paraffin-embedded archived
material. In this talk I will describe the MCC strategy in the general
context of digital PCR, how we have applied it to challenging
clinical samples and how we have now modified the protocol to
derive both multilocus copy-number and sequence information
(including using NGS) from these samples.
Digital PCR or Finding The Needle In A Haystack
Elena Grigorenko
Life Technologies, Inc, United States of America
Digital PCR is a new approach to nucleic acid detection and
quantification, which allows to perform absolute quantification of
targets by directly counting the number of target molecules
partitioned into many individual real-time PCR reactions. The
introduction of OpenArray® Real-Time PCR System to perform
Taqman™ chemistry with digital PCR enable researchers to run
9,000 PCR reactions simultaneously. This approach can be used
for a variety of different applications aimed on detection of rare
events such as quantification of viral load in diluted plasma
samples, rare allele detection and single stem cell transcript
analysis. The examples of each application will be discussed.
OpenArray® platform and its new digital PCR capability not only
extend the portfolio of conventional PCR applications, but also
provide a new, more precise answers to many different biological
questions.
Digital Analysis- a bridge to the Clinic
Jim White
NanoString Technologies, United Kingdom
The nCounter System utilizes molecular barcodes and direct single
molecule imaging to detect and count hundreds of unique
biomarkers in a single reaction. The nCounter assay directly
targets nucleic acid molecules through hybridization, eliminating
the need to purify sample, let alone make cDNA or amplify the
signal. The truly digital nature of quantification provides an
accuracy and reproducibility unmatched with other systems. The
nCounter system can work with a large variety of samples, from
RNA purified from fresh biopsies to crude extracts made from
FFPE material. With a portfolio of off-the-shelf and customdesigned applications in mRNA, miRNA and copy number
analysis, the nCounter system is well suited to a variety of
biological problems. In our presentation we will show how the
CodeSet technology, can construct subtle sample enquires that
can with simple iteration unlock the complexity in the interface
between genome and transcriptome. The simple utility of the
System will help provide an ideal format to empower molecular
medicine by providing a bridge to take nucleic acid biomarkers
from research to the Clinic.
High-throughput real-time PCR approach using the
LightCycler®1536 instrument for genotyping and high
discrimination of E. coli pathogroups responsible for
gastrointestinal diseases
Sabine Delannoy1, Lothar Beutin2, Cédric Woudstra1, Patrick
Fach1
1
French Agency for Food, Environmental and Occupational Health
& Safety (ANSES), France; 2NRL for E. coli, Federal Institute for
Risk Assessment, Germany
Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens responsible for a number of human
gastrointestinal diseases, including watery or bloody diarrhea, and
hemorrhagic colitis. Rapid and specific detection of STEC strains
with high virulence for human has become a priority for public
health authorities. The main virulence factor of STEC is the
production of Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2) or its
variants. STEC strains causing severe disease in humans mainly
possess additional virulence factors such as the outer membrane
protein intimin, essential for the intimate attachment and the
formation of attaching and effacing (A/E) lesions on
gastrointestinal epithelial cells whose genetic determinant is
harboured by the LEE pathogenicity island. In addition to the LEE
locus, several other putative pathogenicity islands (PAIs) have
been identified in STEC strains which present a variable repertoire
of effectors genes that encode potential virulence determinants
(nle, as non-LEE encoded effector). Based on a high-throughput
real-time PCR approach using the LightCycler®1536 instrument a
selection of discriminative nle genetic markers mostly encoding
type III secretion system effector proteins allowed to develop an
assay for specific characterization of the most virulent strains of
STEC. These nle genes were detected at different frequencies in
pathogenic and apathogenic E. coli strains indicating their possible
role in virulence. The presence of these genes may be used to
make a “molecular risk assessment” to predict the virulence
potential of STEC strains. For E. coli O26, these data were
corroborated by a genotyping real-time PCR test based on allelic
discrimination of the arcA (aerobic respiratory control protein A)
gene. Results obtained with the LightCycler®1536 instrument
indicate that a combination of molecular detection and genotyping
tools are highly discriminative to clearly distinguish STEC strains
constituting a severe risk for human health from STEC that are not
associated with severe and epidemic disease. This simple
diagnostic approach might be applicable in hospital service labs or
public health laboratories to test strains isolated from foods or
stools of patients suffering from diarrhea.
NGS: Next Generation Sequencing
Time:
Location:
Session Chair:
Tuesday, 29/03/2011: 2:00pm - 6:00pm
Lecture hall 14
Yuk Ming Dennis Lo, Yu-Hui Rogers
reported a large scale study using this technology. Our results
indicate the fetal trisomy 21 can be detected with a sensitivity of
100% and a specificity of 98% using this technology. We have
also recently shown that in addition to trisomy 21, maternal
plasma DNA sequencing can also reveal the genome-wide genetic
map of the fetus. Thus, massively parallel sequencing is likely to
play an increasingly important role in the future prenatal
diagnostics and monitoring.
Qualitative and Quantitative Analysis of RNA by SOLiD
Next Generation Sequencing
Thomas Rygus
Life Technologies GmbH, Germany
The last years have seen Next Generation Sequencing (NGS) as
an upcoming new method replacing many of the established array
based methods, especially in RNA analysis. Users of RNA
sequencing published in a variety of scientific fields beginning with
quantitative gene expression analysis up to structural analysis of
RNA and allele specific expression; also combinations of several
NGS techniques like small RNA sequencing and ChIP Seq have
been shown. Meanwhile an established method, quantitative RNA
analysis by SOLiD now allows the use of internal RNA controls
making sure that a reliable standardization is possible.
The biggest challenges with resequencing methods have been
automating the complete process and improving the turnaround
time for smaller amounts of samples without losing cost efficiency.
In this presentation we will show an overview about the newest
technical developments of the SOLiD system and workflow
together with an overview about the possible applications in RNA
analysis.
Analysis of circulating nucleic acids (CNA) using NGS
technologies
Bertram Brenig1, Julia Beck2, Ekkehard Schütz2, Howard B
Urnovitz2
1
University of Goettingen, Germany; 2Chronix Biomedical GmbH,
Germany
Next generation sequencing technology provides significant
promise in providing personalized medicine in the 21st century. A
recent report has clearly demonstrated in a small number of
patients that unique chromosomal rearrangements identified by
next generation DNA sequencing can be detected in blood by
PCR. Nevertheless significant barriers to successful translation of
this advancement to clinical medicine exist that include knowledge
of unique mutational lesions for each neoplasm and their fate
during clonal expansion. We have used an alternative approach
that is dependent on next generation sequencing of serum DNA
and DNA motif identification and assembly (Mass Sequencing and
Assembly or MSA) technology that requires no prior knowledge of
specific mutational events in diseased tissue. We have
successfully applied MSA technology to invasive breast carcinoma
and neurodegenerative diseases of animals and humans. We
believe this alternative approach provides an opportunity for
translation of complex basic research data to personalized
medicine.
Noninvasive prenatal diagnosis using fetal DNA in
maternal plasma: from digital PCR to next-generation
sequencing
Improved library quantification for High Throughput
Sequencing at the Wellcome Trust of Human Genetics
Oxford
Yuk Ming Dennis Lo
The Chinese University of Hong Kong, Peoples Republic of China
Paolo Piazza
Wellcome Trust Centre for Human Genetics, United Kingdom
Prenatal diagnosis is now an established part of modern obstetrics
care. However, conventional definitive methods for prenatal
diagnosis, such as amniocentesis, are typically invasive and
constitute a risk to the fetus. In 1997, our group reported that cellfree fetal DNA is present in the plasma of pregnant women. This
technology has initially been used to detect paternally-inherited
fetal genetic characteristics, e.g. sex and blood group genes that
are absent in the mother. In 2007, our group reported the use of
digital PCR for the precise quantitative analysis of fetal DNA in
maternal plasma. We demonstrated the theoretical and practical
basis for single molecule analysis for the noninvasive prenatal
diagnosis of trisomy 21. We have since demonstrated that
massively parallel sequencing is a robust method for the
noninvasive detection of fetal trisomy 21 and have recently
Recent advancements in sequencing technologies (Next
Generation Sequencing) developed for the Illumina and Life
Technology platforms have driven down significantly the costs per
base sequenced. Lower costs coupled with significant
improvements in data quality have opened the door to sequencing
projects that had previously been far too expensive to complete.
As a result we are witnessing an increasing demand for high
quality libraries. Significant effort has been invested at the
WTCHG in improving the library preparation workflows through the
introduction of new equipment and automation to remove the
identified process bottlenecks. Adequate quality control of each of
the many different type of libraries produced in our lab is crucial to
the success of the projects that we support. In order to maximize
the data output and quality, cluster generation on Illumina
platforms requires accurate quantification. Alternative kits for
library quantification were evaluated on a Mx3005 qPCR
instrument. The performance of the QPCR NGS Library Quant Kit
was compared to that of other SYBR® Green based kits. Since we
have introduced the Agilent qPCR instrument and reagents in our
workflow, we observed that approximately 85% of the library
sequenced at the WTCHG produced cluster densities within 20%
from the expected value. Consumables for sequencing or unusual
library types have been often the cause for suboptimal cluster
generation. All the changes and improvements introduced in our
lab have contributed to lowering the costs and accelerate the
turnaround time for projects due to reduced failure rates, however,
the rapid and continuous changes in technology will require
constant adjustments to fully utilize it.
Application of the Next Generation Sequencing
Technologies at JCVI
Yu-Hui Rogers
J. Craig Venter Institute, United States of America
I will give an overview of the JTC research areas and how the next
generation sequencing technologies are being applied to support
these activities. Specifically, I will discuss the sequencing
technologies that are in use at JCVI including 454, Illumina and
SOLiD, their capabilities and how we utilize different sequencing
strategies for different applications. I will also give an overview of
the third generation sequencing technologies, including several of
the single molecule sequencing technologies that are in
development and the potential impact of these technologies on
genomic research.
The use of qRT-PCR and high-throughput
transcriptomics for biomarker development
Michael W Pfaffl, Irmgard Riedmaier, Heinrich HD Meyer
Physiology Weihenstephan, TUM, 85354 Freising, Germany
The detection of growth promoting agents is a central topic in
human doping surveillance and agricultural meat production. In
the focus are the discovery and early verification of the illegal drug
application, e.g. hormone cocktails or new xenobiotic growth
promoters. In routine screening, the residues of all known growth
promoters are detected by immuno based assays or
chromatographical methods in combination with mass
spectrometry. To overcome the detection by these routine applied
screening methods, in the previous years new xenobiotic drugs
and application techniques were adapted. To enable a future
efficient tracing of misused anabolic substances it is necessary to
develop new screening and detection technologies for a broad
range of illegal hormones, including newly designed xenobiotic
anabolic agents. To achieve this, many innovative technologies
have been described for biomarker discovery and validation. This
talk provides an overview of the latest transcriptomics based
technologies in biomarker discovery after illegal hormone
application. These –omics technologies can provide essential
information on the mRNA expression of hormone regulated genes
across multiple tissues, organs, treatment stages, and
experimental trials. It highlights recent gene expression results on
mRNA and microRNA level derived from quantitative RT-PCR and
next generation sequencing. On the basis of the expression
results reliable candidate biomarker were selected. To verify if
these gene expression changes could act as first valid biomarkers
bio-statistical methods were applied. Principal Components
Analysis and Hierarchical Clustering were successfully used to
demonstrate the potential of the transcriptomic approach for the
development of a new screening system and to introduce new
gene expression biomarkers.
Deep sequencing as diagnostic tool for highly
pathogenic viruses
Aleksandar Radonic, Andreas Kurth, Wojtek Dabbrowski,
Andreas Nitsche
Robert Koch Institute, Germany
With the advent of the real-time PCR nucleic acid-based
diagnostics has become the gold standard for the identification of
viral and bacterial pathogens in clinical as well as in environmental
samples. Real-time PCR is fast, reliable, and due to the
application of specific “probes” it offers an additional level of
specificity. Because of this pronounced specificity, however, PCRbased techniques may often fail to detect new or emerging
pathogens with differing or so far unknown genetic information.
While multiplex PCR systems are a convenient approach to
specifically detect a wider variety of pathogens in one reaction
vessel, generic PCR-based amplification systems offer a more
open view and can even facilitate the detection of new pathogens.
Compared to electron microscopy, which provides an actual
diagnostic open view with serious restrictions regarding the
detection limit, recently metagenomic approaches based on
massively parallel sequencing techniques have promised to be a
more sensitive valuable tool for the detection of unknown
pathogens. Since it is technically possible to gain sequence
information of all pathogens present in a particular sample, the
most challenging task is to identify the sequences of interest in the
bulk of sequence data obtained by only one sequencing run. In
this presentation the benefits and drawbacks of next generation
sequencing as diagnostic tool will be discussed in comparison to
conventional methods of virus detection.
High Resolution Transcription Profiling using NextGeneration-Sequencing identifies Diagnostic Markers
for Malignancy
Ralf Horres1,3, Björn Rotter1, Peter Winter1, Michalski
Christoph2, Friess Helmut2, Jüngling Ruth1,3, Günter Kahl3,
Nicolas Krezdorn1, Klaus Hoffmeier1, Jörg Kleeff2
1
GenXPro GmbH, Germany; 2Chirurgische Klinik und Poliklinik,
Klinikum rechts der Isar, TU München; 3Goethe Universität
Frankfurt, Institut für Molekulare Biowissenschaften
Since up to 95% of all transcripts are expressed at low levels,
these rare transcripts are extremely important for a cell‘s fate and
function. A SuperTag Digital Gene Expression (ST-DGE) Profiling
approach was established and applied for the analysis of
malignant potential of pancreatic cancers. In contrast to
microarrays ST-DGE is more reproducible, detects higher dynamic
ranges of gene expression, discovers natural antisense transcripts
and also counts rare transcripts, which are obscured in a
microarray’s background signal. The highly reliable gene
expression signatures allow choosing a subset of diagnostic
transcripts converting the assay to a more clinically friendly PCRbased assay. This study aimed at analyzing processes underlying
the tendency towards malignancy from chronic pancreatitis (CP) to
benign intraductal papillary mucinous tumor (IPMT) to highly
malignant ductal adenocarcinoma of the pancreas (PDAC). STDGE generated a complete survey of poly-adenylated transcripts
in excised normal, CP, IPMTs and PDAC pancreatic tissues. Both
the sense (s) and antisense (as) transcriptomes of CP and IPMT
were significantly different from normal pancreas, and all three
deviated from the transcript patterns characteristic for the
tendency to malignancy, finally manifested in PDACs. A subset of
these transcripts is now tested as prognostic and diagnostic qRTPCR-based markers for malignancies in pancreatic tissue.
GenXPro, est. 2005, is a service provider for the analysis of
nucleotide-based information content of any biological sample.
The service portfolio includes the best available, focused and
sensitive techniques, mostly using next generation sequencing,
delivering qualitative and quantitatively most accurate results, at
lowest possible costs. GenXPro offers full-service, “from tissue to
data” including most suited bioinformatics approaches for the
individual next generation data handling. Our data can be
conveniently analyzed using both our own “analysis suite” and /or
excel. Our analyzes include quality assessment, quantification,
transcriptome mapping, Genome mapping, Gene Ontology
analysis, plots , etc. to ease the analysis and publications. Our
technical service portfolio is headed by our patented „SuperSAGE“
and „SuperTag Digital Gene Expression“ (ST-DGE) Profiling, for
ultra deep transcriptomics studies, but also includes RNAseq,
normalization & sequencing of cDNA, microRNA analysis,
epigenetics, copy number variation detection and genotyping as
well as qPCR service. GenXPro is involved in the development of
several diagnostic genetic markers for human diseases and in
plant and animal breeding.
RNAi - microRNA - siRNA Applications
Time:
Location:
Session Chair:
Tuesday, 29/03/2011: 8:30am - 12:00pm
Lecture hall 15
Jo Vandesompele,
Jan Hellemans
Development of an ultra-high throughput long noncoding RNA qPCR screening system
Jan Hellemans1, Pieter Mestdagh2, Steve Lefever2, Barbara
D'haene1, Filip Pattyn2, Frank Speleman2, Jo Vandesompele1
1
Biogazelle, Ghent, Belgium; 2Ghent University, Ghent, Belgium
Long non-coding RNAs (lncRNA) are an underexplored class of
non-coding RNAs and have been shown to be implicated in health
and disease. They constitute a new class of biomarkers with
disease associated lncRNA signatures yet to be discovered. In
addition, they open up a new approach to understand the function
and organization of the genome. The lack of a high-throughput
platform to detect and quantify lncRNAs has hampered their study
so far. To address this, we developed a new platform for ultra-high
throughput RT-qPCR analysis of long non-coding RNAs. In the
pilot phase, we designed qPCR assays for +1000 lncRNAs based
on public sequence databases and lincRNA chromatin signatures.
All assays were designed using state-of-the-art in silico quality
controls, followed by extensive empirical validation according to
MIQE guidelines. Validation of this first set in relevant biological
model systems demonstrated excellent sensitivity and specificity
of the technology. Data processing was done using the qbasePLUS
software with an improved global mean normalization procedure to
better remove technical variation. In conclusion, we have
successfully completed the first stage of the development process
of an ultra-high throughput and low-volume RT-qPCR platform for
the quantitative detection of lncRNAs. The tool offers a unique
way to investigate the expression patterns of lncRNAs in health
and disease.
Towards blood-based cancer screening using LNA™enhanced microRNA qPCR
Ditte Andreasen, Niels Tolstrup, Jacob Ulrik Fogh, Søren
Jensby Nielsen, Kim Bundvig Barken, Adam Baker, Peter
Mouritzen
Exiqon, Denmark
microRNAs (miRNAs) constitute a recently discovered class of
small RNAs (typically 21-23 nt) that function as post-transcriptional
regulators of gene expression. Current estimates indicate that
more than one third of the cellular transcriptome is regulated by
miRNAs, and miRNAs have been proposed to be master
regulators of cellular state. Indeed, changes in miRNA expression
patterns have been associated with disease states, including a
diverse array of human cancers. Furthermore, the high stability of
miRNA in common clinical source materials (e.g. FFPE blocks,
plasma, serum, urine, saliva, etc.) and the ability of miRNA
expression profiles to accurately classify disease states have
positioned miRNA quantification as a promising new tool for a
wide range of diagnostic applications. To facilitate discovery and
clinical transfer of miRNA-based diagnostic markers, we
developed the miRNome-wide LNA™-based miRCURY LNA™
Universal RT microRNA PCR platform with unparalleled sensitivity
and robustness. Using a specialized design algorithm (available
as a web-tool for custom design of RNA species < 30 nt), we have
designed assays for human and rodent miRNAs. The platform
uses only 40 ng total RNA in just a single RT reaction to profile
>700 human miRNAs in two predefined 384 well plates and thus
allows high-throughput profiling of miRNAs from important clinical
sources without the need for pre-amplification. Following
screening, it is possible to choose a sub-set of miRNA assays for
validation to be formatted in 96- or 384-well plates using our
Pick&Mix platform. The high sensitivity of the assays makes it
possible to do high quality microRNA expression profiling in
samples that contain very little total RNA, such as sections of
formalin fixed paraffin-embedded samples (FFPE) and blood
serum and plasma. Results will be presented demonstrating the
application of the new qPCR method in the profiling of miRNAs
from plasma as part of our efforts to develop molecular diagnostic
tests for treatment selection, diagnosis and monitoring of cancer.
We will also show results demonstrating how selection of a proper
set of miRNA biomarkers in a Pick&Mix plate format can give
precise classification of disease progression.
MicroRNA Target Validation with MISSION® 3'UTR
Lenti GoClone™ and Human MicroRNA Mimics
Nikos Hontzeas
Sigma Aldrich, United States of America
MicroRNAs (miRNAs) may regulate several hundreds of genes to
control a cell’s response to developmental and environmental
signals. The validation of potential target genes is essential in
determining a miRNA’s role and function in these pathways. Here
we use MISSION® 3'UTR Lenti GoClone™ and Human MicroRNA
Mimics to demonstrate validation of known and conserved miRNA
targets. Known targets of hsa-miR-29b (MCL1), hsa-miR-124
(MAPK14), and hsa-miR-373 (LATS2), and targets of conserved
miRNAs hsa-miR-373 (RBL2) and hsa-miR-10a, (HOXD10), were
down-regulated by their respective mimic. These result
demonstrate that the use of MISSION® 3'UTR Lenti GoClone™
with Human MicroRNA Mimics is a viable option for miRNA target
validation.
Impact of different normalization strategies on miRNA
profiling experiments
Swanhild Meyer1, Sebastian Kaiser2, Carola Wagner3,
Christian Thirion4, Michael Pfaffl1
1
Physiology Weihenstephan, Technische Universität München,
Germany; 2Department of Statistics, Ludwig-MaximiliansUniversität München, Germany; 3IMGM Laboratories GmbH,
Martinsried, Germany; 4Friedrich-Baur-Institute and Department of
Neurology, Ludwig-Maximilians-Universität München, Germany
Selection of the normalization strategy has significant impact on
the detection of differentially expressed microRNAs (miRNAs)
from profiling experiments. Normalization techniques currently in
use for miRNA profiling analyses are in analogy to mRNA data
processing or are specifically modified and developed for miRNA
data. Studies evaluating the impact of normalization on miRNA
profiling experiments have focused on different profiling platforms
or the comparison of normalization techniques within one platform.
Here, we investigated the impact of seven different normalization
methods (reference gene index, global geometric mean, quantile,
invariant selection, loess, loessM, and generalized procrustes
analysis) on intra- and inter-platform performance of a one-colour
hybridization-based platform (AGL array) and a multiplex RTqPCR platform (TLDA) and validated results by singleplex RTqPCR assays.
RNAi and gene expression profiling as a tool in cancer
research
Michelle Plusquin1, An-Sofie Stevens1, Katrien De Mulder2,
Frank Vanbelleghem1, Peter Ladurner2, Ann Cuypers1, Tom
Artois1, Karen Smeets1
1
Hasselt University, Belgium; 2University of Innsbruck
As innovative model organisms in stem cell research, the flatworm
species Schmidtea mediterranea and Macrostomum lignano were
used to characterize carcinogenic events. To discover potential
biomarkers, RNAi knockdown and qPCR profiling were combined.
Both cancer inducers as well as blockers were used and
differential expression was compared. Underlying mechanisms
involve oxidative stress related processes resulting in altered gene
expression profiles in function of (stem) cell proliferation. As heat
shock proteins (HSP) appear to play a significant role herein, the
effect of a chemotherapeutic HSP blocker was evaluated. The
value of both RNAi experiments as well as gene expression
profiling is highlighted in this presentation, and an overview of
carcinogenic stress mechanisms and their interactions is given.
Absolute and relative quantification of placental
specific microRNAs in maternal circulation in placental
insufficiency related complications
Ilona Hromadnikova1, Katerina Kotlabova1, Jindrich Doucha2,
Klara Dlouha3
1
Third Faculty of Medicine, Charles University Prague, Czech
Republic; 2Clinic of Obstetrics and Gynecology, University Hospital
Motol, Prague, Czech Republic; 3Institute for the Care of the
Mother and Child, Prague, Czech Republic
The primary goal of our study has been to identify placental
specific microRNAs present in maternal plasma differentiating
between normal pregnancies and non-pregnant individuals. The
selection of appropriate pregnancy associated microRNAs with the
diagnostical potential was based on following criteria: (1) detection
rate of 100 % in full-term placentas, (2) detection rate of above 67
% in maternal plasma throughout gestation (at least 4 positive
wells out of 6 tested wells) and (3) detection rate of 0 % in whole
peripheral blood and plasma samples of non-pregnant individuals.
Initially, we tested microRNAs (miR-34c, miR-372, miR-135b and
miR-518b) which had been previously identified as pregnancyassociated miRNAs. Additionally we selected 16 other highly
specific placental miRNAs (miR-512-5p, miR-515-5p, miR-224,
miR-516-5p, miR-517*, miR-136, miR-518f*, miR-519a, miR-519d,
miR-519e, miR-520a*, miR-520h, miR-524-5p, miR-525, miR-526a
and miR-526b) from the miRNAMap database. Seven microRNAs
(miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525
and miR-526a) were newly identified as pregnancy associated
with diagnostic potential. Further, we examined if extracellular
placental specific microRNAs (miR-520a*, miR-520h, miR-525 and
miR-526a) can differentiate pregnancies with placental
insufficiency related complications from normal ones. Absolute
and relative quantification of placental specific microRNAs (miR520a*, miR-520h, miR-525 and miR-526a) was determined in 50
normal pregnancies, 32 complicated pregnancies (21
preeclampsia with or without intrauterine growth retardation and
11 IUGR) and 3 pregnancies at various gestational stages who
later developed preeclampsia w/o IUGR and/or IUGR using realtime PCR and the comparative Ct method relative to ubiquitous
miR-16. Both quantification approaches revealed significant
increase of extracellular placental specific microRNAs levels over
time in normally progressing pregnancies, however did not
differentiate between normal and complicated pregnancies at the
time of preeclampsia and/or IUGR onset. Nevertheless, significant
elevation of extracellular microRNAs was observed during early
gestation (within 12th to 16th weeks) in pregnancies with later onset
of preeclampsia and/or IUGR. To avoid instability of extracellular
microRNAs in maternal plasma, prenatal monitoring should be
performed using only the samples with the storage life below two
months. Early gestation extracellular microRNAs screening may
differentiate between normal pregnancies and those who will later
develop placental insufficiency related complications.
Acknowledgements: This work was supported by grant projects MSM
0021620806 and GAUK 260707/SVV/2010.
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New qPCR Applications and Method Optimisation
Time:
Location:
Session Chair:
Tuesday, 29/03/2011: 2:00pm - 6:00pm
Lecture hall 15
Heinrich HD Meyer, Robert Loewe
Quantitative Immuno-PCR based on Imperacer:
Technology, Platform & Analytical Applications
Jan Detmers, Mark Spengler, Michael Adler
Chimera Biotec GmbH, Germany
While conventional ligand-binding assay formats like ELISA or
ECL provide sufficient sensitivity in biological matrices to support
many bio-analytical projects, a number of ligand-binding assay
requires ultra-sensitivity. Immuno-PCR (IPCR) combines the high
specificity of the antibody-antigen interaction with the exponential
signal amplification by PCR as the most sensitive detection
method in molecular biology, to an ultra-sensitive ligand-binding
assay format. Due to exponential signal generation, assay
sensitivity and broad detection range are the main advantages of
IPCR over conventional ligand-binding assays. Here, we present a
range of case studies, making use of Imperacer®, an IPCR based
immunoassay system, in combination with tailored sample dilution
procedures to enhance assay robustness and precision by
minimizing interfering effects from the sample matrix, endogenous
counterparts of the analyte or the biotherapeutic drug candidate in
immunogenicity assessment. This approach has successfully
been demonstrated in fields like PK/PD, Biomarkers, Replacement
Therapy, Monitoring of Mucosal Vaccination, Virus Load Detection
and Anti-Drug Antibody Testing.
Quantitative Assessment of Chromatin Structure:
Analysis of Epigenetically Regulated Gene Promoters.
Francisco Bizouarn
Bio-Rad Laboratories Inc, United States of America
qbasePLUS: data analysis lunch seminar
Time:
Location:
Session Chair:
Tuesday, 29/03/2011: 1:00pm - 2:00pm
Lecture hall 15
Barbara D'haene
Jan Hellemans
qbasePLUS 2.0: the next generation of real-time PCR
data-analysis software
Biogazelle, Belgium
Biogazelle invites you to a 45 minutes live demonstration during
which we will showcase qbasePLUS 2.0, a major upgrade of the
most powerful, flexible, and user-friendly real-time PCR dataanalysis software.
First, we will walk you through the analysis of a small qPCR
experiment highlighting the need for quality control while handling
qPCR data. Tips and tricks provided during this demonstration will
Epigenetic processes, such as DNA methylation and histone
modification, control gene expression by altering chromatin
structure. Genes that are actively transcribed are associated with
“open” or “accessible” chromatin regions; genes that are
transcriptionally silent are often in “closed” or “inaccessible”
chromatin. We present a new assay, termed the EpiQ chromatin
assay, that quantitatively assesses chromatin structure in cultured
cells. The assay is novel, easy to perform, quantitative and
produces results on the day of cell harvest. We validated this
assay by analyzing 15 genes in four human cancer cell lines (4
housekeeping genes and 11 genes that are epigenetically
regulated in human cancer). We observe an excellent correlation
between the level of gene expression and the chromatin structure
of the corresponding promoter. Importantly, our data indicates that
gene promoters exist not just in “open” and “closed” chromatin
states, but also exist in intermediate states of chromatin
accessibility which act to fine-tune the level of gene expression.
The EpiQ chromatin assay compliments and extends existing
epigenetic assays and allows researchers to gain novel insights
into mechanisms of gene regulation.
Quantification of specific mutation with HRM for
regions of interest
Robert Loewe
GeneWake GmbH, Germany
HRM has been implemented on numerous instruments with many
chemistry options. Despite this fact optimization and quantification
of e.g. mutational status has not yet reached most of the labs.
Therefor a comparison of instruments and different chemistry was
done. As a model system the SNP responsable for lactose
intolerance (rs4988235) was used to find optimal amplicon length
(tested from 60 - 653bp). Also we tested the possibility to calculate
the mutational percentage for diagnostic purposes. The outcome
presented a potential solution for quantitative HRM with the
possible usage in screening minute sample material for mutational
status.
Quantitation of Rare Undifferentiated hESCs by
Methylation-Specific CastPCR (MeS castPCR)
Jason Gioia, David Deng, Jeff Rosner, Caifu Chen
Human embryonic stem cells (hESCs) retain the remarkable ability
to differentiate into multiple different tissues and to self-renew in
vitro. These remarkable abilities make hESCs very attractive to
both scientific and clinical communities because they are
potentially a renewable source of a wide variety of human tissues
which can be used for regenerative medicine, drug discovery and
toxicity testing. However, during the cell type-specific
differentiation of hESCs, a small fraction of undifferentiated hESCs
frequently remain mixed with the differentiated cells. These
undifferentiated hESCs pose serious risk by forming tumors after
transplantation into patients. Therefore, it is important to detect &
quantify rare, undifferentiated hESCs in therapeutic stem cell
products. Based on distinct methylation patterns between hESCs
and their differentiated cells in a set of genes, here we propose a
new technology called methylation-specific castPCR (MeS
castPCR) for accurate and sensitive quantification of
undifferentiated hESCs. This new technology will bridge the gap in
hESC product QC and then overcome this major obstacle in stem
cell therapy. CastPCR combines allele-specific TaqMan qPCR
with allele-specific minor grove binder (MGB) as blockers to
suppress effectively non-specific amplification from the wild type
allele. We have successfully designed and validated castPCR
assays for ~100 SNPs or InDels. Results demonstrate that
castPCR not only maintains wide dynamic range, high sensitivity,
and reproducibility of TaqMan assays but also is able to detect
one mutant in 100,000,000 wild-type molecules. Recently, Laurent
et al. (2010) and Lister et al. (2009) presented a whole-genome
comparative view of DNA methylation using bisulfite sequencing of
hESCs and various differentiated cells. Based on their findings, we
have identified genes and genomic DNA regions such as HOXB6
and others which show distinct DNA methylation patterns between
hESCs and differentiated cells. These regions could be used as
methylation markers to distinguish between hESCs and their
differentiated (somatic) cells. It is important to note that DNA
regions with 0% methylated in hESC and 100% in differentiated
cells are mostly desirable due to incomplete bisulfate conversion.
MeS castPCR is aimed to detect converted CpG sites which are
used to estimate % of hESC contamination in therapeutic cell
products. Our results suggest that MeS castPCR is capable of
detecting hESC contamination as little as 0.01% in differentiated
stem cell therapy product during clinical trials.
PrimeTime® Pre-designed qPCR Assays using ZEN™
Internal Dark Quencher Chemistry
Scott D. Rose
Integrated DNA Technologies, United States of America
Well-designed qPCR assays require the careful consideration of
primer placement, specificity, avoidance of single nucleotide
polymorphisms, oligonucleotide interactions and accurate melting
temperature calculations. Using pre-designed assays shifts the
burden of assay development from the researcher to the
manufacturer. Unfortunately, some of the pre-designed assay
sets commercially available today were built using outdated
databases and primer design was done with older, less accurate
Tm algorithms. Even worse, some suppliers do not provide
sequence information to the end user that would enable the
researcher to evaluate the reagents in the context of their specific
requirements or for use in publication. IDT now offers fully
guaranteed PrimeTime® Pre-designed qPCR assays (for all genes
in the human, mouse, and rat genome) that avoid cross reactivity
within that genome, known single nucleotide polymorphisms,
primer interactions, and gives full disclosure of all sequence
information as recommended by MIQE guidelines. By including
ZEN double-quenched probes, these assays offer sensitivity to 10
copies or lower with the added security of knowing precise probe
location with full sequence disclosure.
PrimerXL: high-throughput assay design for qPCR and
amplicon based NGS
Filip Pattyn, Steve Lefever, Frank Speleman, Jo
Vandesompele
Ghent University, Belgium
Primers are the cornerstone of many modern-age high-throughput
nucleic acid analysis techniques ranging from real-time
quantitative PCR (qPCR) to amplicon generation for nextgeneration sequencing. Various tools are currently available for
automated primer design, but most lack a thorough downstream
evaluation. A proper assessment of the multiple aspects affecting
a primer assay's efficiency is a laborious and repetitive work
involving manual interactions with different software applications.
We automated the primer design process from top to bottom into a
processing pipeline combining experimentally optimized design
guidelines with a multi-faceted primer evaluation. This evaluation
includes checking for the presence of SNPs in the primer
annealing sites to avoid sample dependent amplification
efficiencies, evaluating secondary structures in the primer
annealing sites hampering efficient primer annealing and testing
the primer specificity which results in primers having an good
overall efficiency. The pipeline is customizable, making it possible
to design primers fitting specific needs. In addition to the
development of standard qPCR-primers, the pipeline allows to
design primers targeting a specific transcript variant or a subset of
transcript variants of a single gene or specific gene loci like
putative transcription factor or miRNA binding regions.
Another field in which PrimerXL exceeds concurrent tools is the
design of primers used in the preprocessing steps of amplicon
based next-generation sequencing. Until now, the application of
high-throughput amplicon sequencing has been hindered by the
lack of tools capable in developing efficient tiling primers. To fill
this void, we have expanded our primer design pipeline with a
module to design tiling primers targeting all exonic sequences of a
specific gene. The generated primers produce more equimolar
amplicon sets leading to a cost-efficient and sensitive variation
detection without extra sequencing efforts. The low variation in
amplicon molarity outperforms the widely used array capturing
technology employed to amplify specific target regions.
Natasha Paul
PCR is a widely used scientific tool whose specificity can be
increased by the use of Hot Start technologies. Although many
Hot Start technologies exist, recently developed CleanAmp™
dNTPs are a distinct approach that employs modified nucleoside
triphosphates with a thermolabile protecting group at the 3'hydroxyl. The presence of the protecting group blocks low
temperature primer extension, which can often be a significant
problem in PCR. At higher temperatures, the protecting group is
released to allow for incorporation by the DNA polymerase and
more specific amplification of the intended target. These modified
dNTPs provide comparable performance to other Hot Start
technologies and can be used with thermostable DNA
polymerases to turn a reaction into a Hot Start version. This
thermolabile chemistry can be applied to dNTP analogs such as
dUTP, which is used in UNG decontamination methods, and 7deaza-dGTP, which is used to amplify difficult GC-rich targets. In
addition, further studies have led to the development of 3'protecting groups that deprotect more quickly than the current 3'modification group, allowing these modified dNTPs to be used in
fast PCR. With the evolving chemistry of the hot start dNTPs, the
areas of application benefiting from the versatility and flexibility of
this technology continue to grow.
PCR-Luminex Based Detection of 28 Enteropathogens
1
1
2
1
Mami Taniuchi , Jie Liu , Shihab Sobuz , William Petri ,
Rashidul Haque2, Eric Houpt1
1
University of Virginia, United States of America; 2International
Centre for Diarrhoeal Diseases and Research, Bangladesh
Background: Given the range of potential enteropathogens
implicated in diarrhea, we developed a PCR-Luminex based assay
to detect 28 most common organisms associated with diarrhea.
The enteropathogens included were Cryptosporidium spp.,
Entameoba histolytica, Giardia, Microsporidia, Cystoisospora,
Cyclospora, Ascaris, Hookworms, Strongyloides, Tricuris, EAEC,
EHEC, EIEC, EPEC, ETEC, Astrovirus, Norovirus G1 and G2,
Rotavirus, Sapovirus, Adenovirus, and an internal control. This
panel was used in a prospective study to determine etiology of
diarrhea in Bangladeshi children in the first year of life. Methods:
Specific primers and probes were designed for the organisms
using targets which are conserved. DNA or RNA purified from
stool was amplified using biotinylated primers, followed by
hybridization to amine-modified probes covalently linked to
carboxylated spectrally-distinct microspheres, followed by addition
of streptavidin PE to detect specifically-bound amplicon. Luminex
results are reported as corrected mean fluorescence intensity
(cMFI) normalized to background, where cMFI > 2.5 was utilized
as a “present” call with the exception of Cryptosporidium,
Strongyloides, all EC, and all viruses where cMFI values were 7.3,
9.0, 10.0, and 2.0, respectively. Results: Performance of the
assays yielded 95% to 100% sensitivity and specificity versus the
assays performed via real-time PCR. We then applied the tests in
a prospective study of 147 children from Mirpur, Bangladesh
followed monthly for the first year of life. 83% of the children had
at least 1 episode of diarrhea in the first year of life and 33% had 4
or more. The number of pathogens that were detected increased
as the number of diarrheal episodes increased (e.g., from 1.7 to
2.5 from first diarrheal episodes to 4th or later diarrheal episodes,
P <0.05). Rotavirus, ETEC, EAEC, and were the leading
pathogens detected during the first diarrheal episodes, while
Adenovirus, Shigella, ETEC, EPEC, and Giardia emerged as
common in the later episodes. Conclusion: This Luminex based
assay for the major enteropathogens offers sensitive and specific
detection similar to real time PCR. When applied to field studies in
endemic areas, a singular etiology of diarrhea is difficult to
determine due to the frequency of mixed infections, and multiple
pathogens may be the norm. Pathogens appear to accumulate in
children that develop recurrent diarrhea.
Simple analytical and experimental procedure for
selection of reference genes for qPCR normalization
data
Rodrigo Manjarin1, Nathalie Trottier1, Patty Weber2, James
Liesman1, Nathanael Taylor1, Juan Pedro Steibel1,3
1
Department of Animal Science, Michigan State University, United
States of America; 2Department of Large Animal Clinical Sciences,
Michigan State University, United States of America; 3Department
of Fisheries and Wildlife, Michigan State University, United States
of America
Variation of cellular activity in a tissue induces changes in RNA
and DNA concentration between samples, which may affect the
validity of mRNA abundance of target genes obtained with realtime quantitative PCR (qPCR) analysis. A common way of
accounting for such variation consists of the use of a reference
gene as a normaliser. Programs such as geNorm can be used to
select suitable reference genes, although a large set of genes that
are not co-regulated must be analyzed to obtain accurate results.
The objective of this study was to propose an alternative analytical
protocol to assess the invariance of reference genes in porcine
mammary tissue using mammary RNA and DNA concentrations
as correction factors. Mammary glands were biopsied from 4 sows
on d 110 of gestation (pre-partum), on d 5 (early) and 17 (peak) of
lactation, and on d 5 after weaning (post-weaning). Relative
expression of seven potential reference genes, API5, MRPL39,
VAPB, ACTB, GAPDH, RPS23 and MTG1, and one candidate
gene, SLC7A1, was quantified by qPCR using a relative standard
curve. The response in cycles to threshold at each stage of
lactation was tested using a linear mixed model fitting RNA and
DNA concentration as covariates. Results were compared to those
obtained with geNorm analysis and genes selected by each
method were used to normalize SLC7A1. Quantified relative
mRNA abundance of API5, GAPDH and MRPL39 remained
unchanged (P > 0.1) across stages after correcting with RNA and
DNA concentration, whereas geNorm analysis selected MTG1,
MRPL39 and VAPB as best reference genes. There were no
differences between normalization of SLC7A1 with genes selected
by the proposed analysis protocol or by geNorm. In conclusion,
the proposed analytical protocol and geNorm selected different
reference genes, but SLC7A1 fold changes did not differ when
using genes obtained under either method. These results indicate
that among our set of candidate genes, there is more than one
suitable reference gene. The proposed method, however, has the
strength of allowing testing each potential reference gene
individually and sequentially, so analysis of remaining genes can
be spared as soon as a suitable reference gene is identified.
th
Wednesday 30 March 2011
Pre-PCR: Pre-analytical Steps
Time:
Location:
Session Chair:
Wednesday, 30/03/2011: 9:00am - 12:00pm
Lecture hall 14
Jo Vandesompele, Karl H. Hasenstein
Measurable impact of RNA quality on gene expression
results from quantitative PCR
Joëlle Vermeulen1, Katleen De Preter1, Steve Lefever1, Justine
Nuytens1, Fanny De Vloed1, Stefaan Derveaux1, Jan
Hellemans2, Frank Speleman1, Jo Vandesompele2
1
Ghent University, Ghent, Belgium; 2Biogazelle, Zwijnaarde,
Belgium & Ghent University, Ghent, Belgium
Compromised RNA quality is suggested to lead to unreliable
results in gene expression studies. Therefore, assessment of RNA
integrity and purity is deemed essential prior to including samples
in the analytical pipeline. This may be of particular importance
when diagnostic, prognostic or therapeutic conclusions depend on
such analyses. In this study, the comparative value of six RNA
quality parameters was determined using a large panel of 740
primary tumour samples for which real-time quantitative PCR gene
expression results were available. The tested parameters
comprise microfluidic capillary electrophoresis based 18S/28S
rRNA ratio and RNA Quality Index value, HPRT1 5’-3’ difference
in quantification cycle (Cq) and HPRT1 3’ Cq value based on a
5’/3’ ratio mRNA integrity assay, the Cq value of expressed Alu
repeat sequences, and a normalisation factor based on the mean
expression level of 4 reference genes. Upon establishment of an
innovative analytical framework to assess impact of RNA quality,
we observed a measurable impact of RNA quality on the variation
of the reference genes, on the significance of differential
expression of prognostic marker genes between two cancer
patient risk groups, and on risk classification performance using a
multigene signature. This study forms the basis for further rational
assessment of RT-qPCR based results in relation to RNA quality.
Isolation and Analysis of Circulating Nucleic Acids:
Technical Advances
Martin Horlitz
QIAGEN GmbH, Germany
Circulating DNA fragments and specific messenger RNAs
originating from malignant tumors, a developing fetus, but also
viral or bacterial infections are present in the cell-free nucleic acids
in plasma, serum and other body fluids such as urine. Access to
these nucleic acids for analysis could allow for specific detection
of certain disease states based on a simple blood sample.
Circulating cell-free nucleic acids show distinctive properties: They
are present in plasma and serum mostly as shorter fragments of
less than 500 bp or nt in size. The concentration of such
circulating, cell-free DNA and RNA in a plasma or serum sample is
low (≈1–100 ng/ml) compared to cellular materials and varies
considerably between different individuals. Because of their
fragmented nature and low concentration, circulating nucleic acids
present a particular challenge for efficient extraction/purification
and quantification by qPCR. We present data on solutions for the
following critical problems concerning the analysis of circulating
nucleic acids for research and molecular diagnostic applications:
- Pre-analytical workflow (blood processing) for analyzing
circulating nucleic acids
- Optimization of ccfDNA extraction from plasma samples: low
target concentrations require the efficient ccfDNA enrichment from
larger sample volumes (1-5 ml)
- Use of an internal control to monitor the ccfNA extraction
performance
- Avoiding qPCR Quantification bias when working with
fragmented target nucleic acids
- Assessment of ccfDNA fragment size distribution in plasma
samples by comparing qPCR results based on target amplicons of
different sizes
Furthermore, we will present data on the extraction and qPCRbased quantification of circulating fetal DNA and mRNA from
maternal plasma samples obtained at the end of the first trimester
of pregnancy.
Solid Phase Gene Extraction – Sampling of mRNA
from living systems
Karl H. Hasenstein, Min Chen
Univ. Louisiana Lafayette, United States of America
Understanding gene expression and regulation especially on a
cellular level requires rapid, localized and ideally repeated
sampling of mRNA. Current technologies inevitable rely on the
extraction of tissue, cytoplasm aspiration, or cell lysates, all of
which pose problems because of unspecific sampling,
contamination, and lack of repeatability. These methods are
destructive and do not distinguish between genomic DNA and
RNA. Moreover, extracted mRNA is typically contaminated by
extracted cytoplasm, nuclear DNA, or other compounds, and the
required purification leads to loss of especially low-abundant
mRNA. Biological systems, where all regulatory and response
steps take place in a single cell are thus not accessible to
localized gene expression studies. This predicament lead to the
development of a needle-based mRNA extraction system that
provides high temporal and spatial resolution that allows for
repeated removal of mRNA from living material. This development
(dubbed Solid Phase Gene Extraction, SPGE) can be designed to
hybridize with gene-specific or generic (oligo-dT) sequences. It is
not species-specific and can be employed to access tissue or
single cells. We demonstrate the versatility and validity of this
novel RNA extraction technique by simultaneously profiling nanos
and bicoid mRNA in individual Drosophila eggs. The distribution of
these genes resulted in the same longitudinal distribution as
previously described distribution profiles. The low impact of SPGE
sampling was underscored by the normal development of
repeatedly sampled eggs. Actin isoforms in germinating seedlings
varied in the apical tissue of roots as a function of imbibition and
permits discrimination between newly synthesized and maternal
genes. Gene expression in human tumor tissue suggests that
SPGE has the potential to supplement or eliminate biopsies.
Examination of several genes that are typically used as references
in gene expression studies show that SPGE is more sensitive than
tissue-based extraction and has the potential to function as a selfreferencing system. These data demonstrate the universality of
SPGE as a simple, generic, analytical, and diagnostic procedure.
Impact of basepair mismatch in primer annealing on
qPCR assay performance
Steve Lefever, Pattyn Filip, Speleman Frank, Hellemans Jan,
Vandesompele Jo
Center for Medical Genetics Ghent, Ghent University, Belgium
With the onset of the 1000 Genomes Project that aims to explore
human genetic variation, a multitude of new single nucleotide
(SNP) polymorphisms have emerged. Therefore, the density and
distribution of known variants has increased significantly and
hampers design of primers in regions devoid of SNPs.
Importantly, perfect primer/target complementarity is believed to
be crucial in all samples since the presence of a SNP in the primer
annealing region may affect the result of a quantitative polymerase
chain reaction (qPCR). In the worst case, absence of amplification
of the variant allele has been reported in literature. However, the
impact of such a mismatch in relation to its location in the primer
and local sequence context has not been systematically studied.
To address this important issue, we have designed a study to
systematically and empirically assess the relation between the
presence of a mismatch in the last five 3’ end bases of a PCR
primer and various qPCR parameters, such as efficiency and Cqvalue. The study is based on a constant reverse primer and a set
of nine walking forward primers, located up to four bases up- or
downstream from a starting position, where single base pair
mismatches have been introduced at the 3’ end final base. 36
forward primers and 16 synthetic oligonucleotide templates (good
for 576 distinct and informative match/mismatch reactions) were
designed to prevent secondary structure formation, self-annealing
and primer dimerization. Different qPCR reaction mixes with
variable sensitivity towards primer mismatches are evaluated.
Data analysis is currently being finalized and results will be
presented at the symposium.
Gene Expression profiles from FFPE samples with
improved RNA decrosslinking technology A case
study: Molecular profiling of breast cancer from
formalin-fixed, archival material
Guido Krupp1, Susanne Quabius2, Rolf Jaggi3
1
AmpTec GmbH, Hamburg, Germany; 2Institute for Immunology,
University Klinikum UK-SH, Kiel, Germany; 3Dept. Clinical
Research, University of Bern, Bern, Switzerland
We have developed a novel demodifaction/decrosslinking protocol
for RNA recovery from archival (FFPE) material. The resulting
FFPE RNA quality is superior to RNA obtained with other
commercial FFPE RNA isolation kits: larger RNAs can be
recovered, and RT-qPCR data demonstrate less variability and
lower Cq values. This FFPE RNA is suitable for differential gene
expression measurement by qPCR, high concordance with
parallel RNA samples from fresh-frozen tissues was observed.
Prognosis of breast cancer is determined by clinicopathological
and molecular factors. We developed and validated molecular
scores reflecting the hormone status (ER, PGR, HER2 scores)
and the proliferation status (PRO score) of breast cancer cells.
The scores can be combined to an overall RISK score. Molecular
scores are independent prognostic parameters, they were
validated in postmenopausal patients with estrogen receptor
positive breast cancer. Multivariate analysis revealed that PRO
and RISK scores outperform conventional parameters (histological
grading and Ki-67 labeling index). Molecular scores are based on
routine pathological material, testing can be implemented easily
into routine diagnosis.
DEPArray™: a novel platform to identify, isolate and
collect very rare cells
Pim van der Aar1, Manuela Banzi2
1
Dync B.V., Netherlands, The; 2Silicon Biosystems SpA
The DEPArray A300K system, uses a microelectronic chip
integrating an array of 307,200 tiny electrodes, implementing up to
76,800 moving dielectrophoretic-cages for software-controlled cell
sorting and manipulation. The chip is embedded in a microfluidic
disposable cartridge enabling multiple recoveries of individual or
group of cells, as well as manipulation of cells and/or microbeads
for cell biology experiments.
Cells injected in the chip are trapped in an array of field cages.
Images are then acquired, and cell selected for sorting or
manipulation with the following unique features:
- sort by morphological parameters such as shape, nucleus to
cytoplasm ratio, fluorophores co-localization, thanks to image
based selection
- no contamination from spurious events verifying images
associated to cells selected from the scatter plot or histogram,
including up to 5 fluorescence channels and bright-field
- no need for a-priori thresholds, pick-up only best cells available
in the sample
- sort specific rare-cells, based on relative fluorescence
parameters combined with morphology
- sort cells from a suspension where the total cell count is
extremely low (0-100,000)
DEPArray is compatible with PBS and culture media. This allows
one to manage live cells, in physiological conditions, without the
need for special low-conductivity buffers.
With respect to other cell manipulation techniques such as optical
tweezers, DEPArray™ has the key advantage that
1) cells need not to be in the field of view of the microscope to be
manipulated
2) a massive amount of cells can be trapped and managed stepby-step, deterministically, under software control
These advantages translate in a whole new world of possibilities
beyond sorting, such as for example
- to run experiment of cell-cell interaction with exquisite control on
timing and parallelism of the interactions, or
- to stimulate cell receptors with functionalized microbeads, in a
controlled way
discriminate the two SNP homozygous populations by HRM on a
real-time PCR platform. p53 is currently the most frequently
mutated tumour-suppressor gene in human cancers. A SNP in
codon 72 of p53 results in either an arginine (Arg) or proline (Pro)
residue, and has been demonstrated to affect p53 function. Our
results show that the FRET primer coupled with HRM analysis
reliably enables discrimination between Arg/Pro SNP in 18
peripheral blood sample of cancer patients. The FRET primers
exhibit comparable sensitivity and expansive dynamic range as
compared to SYTO-9 based HRM platform.
Christine Aurnhammer, Maren Haase, Anja Ehrhard, Martin
Häusel, Ingrid Huber, Nadine Muether, Hans Nitschko, Ulrich
Busch, Andreas Sing, Armin Baiker
Bavarian Health and Food Safety Authority (LGL), Germany
New qPCR Applications in Molecular Diagnostics
Time:
Location:
Session Chair:
Wednesday, 30/03/2011: 13:30pm - 4:30pm
Lecture hall 14
Andreas Nitsche
Heinrich HD Meyer
Detection and classification of microorganisms by
combination of qPCR and pyrosequencing
Kati Schroeder, Andreas Nitsche
Robert Koch-Institut, Germany
Microbial and viral typing is usually performed by Sanger DNA
sequencing. Pyrosequencing (PSQ) is a new sequencing-bysynthesis method allowing a fast and reliable detection of
polymorphisms and sequence variations between different
species. Performed in combination with quantitative PCR, PSQ
can verify positive qPCR results and concurrently identify the
pathogen species immediately after the PCR run is finished. To
combine qPCR with PSQ, target regions within the respective
pathogen genome were amplified in parallel by qPCR with one
biotinylated primer for each assay. Positive qPCR results indicated
the presence of a pathogen. Additionally, biotinylated DNA strands
of PCR products were separated and purified on Streptavidincoated beads and hybridized to a sequencing primer. Subsequent
PSQ generated a pyrogram with a specific sequence that allowed
classification of the pathogen by local BLAST search. As different
qPCR and corresponding PSQ assays can be easily performed in
parallel, a broad range of pathogens can be detected rapidly
allowing accurate and reliable microbial and viral diagnostics in
samples of unknown infectious origin.
FRET primer for single nucleotide polymorphism (SNP)
genotyping
Choy Len Fong1, Fiona Ng1, Kanaga Sabapathy2, Kim Halpin1
1
Life Technologies, Singapore; 2Laboratory of Molecular
Carcinogenesis, National Cancer Centre, Singapore
Single nucleotide polymorphisms (SNPs) have become one of the
most important objects of medical research. They have the
potential to identify new drug targets, explain individual differences
in the effectiveness of drugs, and susceptibility to disease.
However the identification of SNP profiles by high resolution melt
(HRM) is currently limited. The goal of this study was to develop
an accurate HRM assay which employs the use of fluorescence
resonance energy transfer (FRET) primers for identifying SNPs. A
FRET primer is internally labeled with a fluorescence dye at a
thymine (T) nucleotide. A Fret primer assumes a randomly coiled
structure as a single stranded primer, resulting in the reporter dye
and quencher being in close proximity, hence quenching
fluorescence. Upon Fret primer extension, a rigid duplex forms,
resulting in reporter dye and quencher separation and hence
increased fluorescence which is ideal for real-time PCR
quantification and melt curve study. In this study, we describe the
feasibility of using a Fret primer for genotyping SNPs with HRMbased genotyping, offering an alternative to an intercalating dye
(i.e. SYTO-9)-based platform. A Fret primer targeting the p53
codon 72 SNP site was designed and tested for the ability to
Background: Viral vectors based on adeno-associated virus
serotype 2 (AAV2) constitute promising tools in human gene
therapy. The inverted terminal repeats (ITRs) within the viral
genome are the only cis-acting viral elements required for
functional AAV2 vector generation and constitute the lowest
common denominator of all AAV2-based vectors. However, so far,
no PCR-based method for the detection and quantification of
AAV2-ITRs could be established due to their extensive secondary
hairpin structure. Current PCR-based methods are therefore
predominantly targeting vector-encoded transgenes or regulatory
elements. Methods: We established an AAV2-ITR sequencespecific quantitative PCR (qPCR) method. Primers and BBQlabeled probe are located within a particular subregion of the ITR
sequence and have been designed to detect wild type AAV2 and
AAV2-based vectors. Results: This method is suitable for the
evidence of both, single-stranded (genomic) DNA derived from
AAV2 vector particles and double-stranded DNA derived from
producer plasmids. The linear dynamic range could be shown for
102 to 107 copies and the detection limit determined as 50 copies.
A practical approach for the analysis of putative cross reactivities
against closely related AAV serotypes utilizing synthetic oligo
nucleotides revealed some cross reactivity against orthologous
sequences of AAV1, 3, 6 and 7 but not against AAV4, 5, 8 and 9.
For AAV vector production adenovirus type 5 (Ad5) is often used
in terms of its helper virus properties. Therefore, we further
investigated the specificity of our qPCR method using Ad5-DNA
and could prove the method to result in no detectable cross
reactivity with Ad5. Conclusion: This method comprises the first
qPCR system facilitating the detection and quantification of AAV2
-ITR sequences. Since this method can be applied for all AAV2based vectors in a “one for all”-based manner, it will significantly
simplify AAV2 vector genome titrations in the future.
Cost-efficient detection of Mycobacterium avium
subsp. paratuberculosis in faeces by quantitative real
time PCR and development of a predictive model for
fighting the disease
Petr Kralik1, Iva Slana1, Alena Kralova1, Vladimir Babak1,
Robert H. Whitlock2, Ivo Pavlik1
1
Veterinary Research Institute, Czech Republic; 2University of
Pennsylvania, School of Veterinary Medicine, PA, USA
Mycobacterium avium subsp. paratuberculosis (MAP) causes
losses in ruminants especially in cattle. The conventional control
programmes are based on the traditional culture that suffers from
the long time required for the incubation. In this study we have
aimed on two main issues. Firstly, we have developed and
optimised a reliable and cost-efficient DNA isolation procedure
from faeces that could be coupled with previously developed
IS900 and F57 quantitative real time PCR (qPCR) for the MAP
detection. To determine the quantity of MAP as precise as
possible, the recovery of MAP DNA from the spiked faecal
samples was established. It ranged from 29.1 to 102.4% of the
input amount of MAP with median 37.9%. The limit of detection
was determined to be 1.03 × 104 for F57 qPCR and
6.87 × 102MAP cells per gram of faeces for IS900 qPCR,
respectively. The developed technique for DNA isolation was
coupled with IS900 qPCR and compared to traditional MAP
culture using a cohort of 1906 faecal samples examined from 12
dairy cattle farms in our laboratory. From those 1906 original
faecal samples, 875 were positive by IS900 qPCR and 169 by
culture. None of the culture positive samples was negative by
IS900 qPCR. This data facilitated development of a predictive
model capable of estimating the probability of being culture
positive by estimating the absolute number of MAP per gram of
faeces as determined IS900 qPCR without performing the culture.
This work was supported by the EC (ParaTBTools), the Ministry of
Education, Youth and Sports of the Czech Republic “AdmireVet” (CZ
1.05/2.1.00/01.0006 and No. ED 0006/01/01) and the Ministry of Agriculture
of the Czech Republic (Grants Nos. MZe0002716202 and QH81065).
Development of a novel duplex real-time PCR assay
for plasma DNA quantification and its comparison to
other methods
Shiyang Pan, Dan Chen, Peijun Huang, Bing Gu, Fang Wang,
Jian Xu, Chun Zhao, Yongqian Shu, Di Yang
The first affiliated hospital with Nanjing medical university, China,
Peoples Republic of
BACKGROUND: It has long been known that plasma DNA is
present in healthy and diseased individuals and many
investigators have been attracted to this emerging field. However,
a direct comparative analysis of the abundant plasma DNA data
available is often prevented by variability in sample storage,
sample processing, and protocols for DNA extraction. Therefore, a
more accurate and stable assay with lower uncertainty is needed
in conjunction with a multi-center study. METHODS: In this study,
a duplex real-time PCR assay with an internal control was
developed as a novel method for quantification of plasma DNA.
The detect limit, precision, accuracy and stabilitity of this novel
assay was then evaluated and compared to other two popular
plasma DNA quantification methods by real-time PCR with
external standards and by direct fluorescent PicoGreen staining.
Furthermore, the plasma DNA concentrations of 1,187 healthy
Chinese adults have been determined by the novel assay.
RESULTS: The detect limits of novel duplex real-time PCR, realtime PCR with external standards and PicoGreen assay were 0.1
ng/ml (R2 = 0.96, P < 0.0001), 0.1 ng/ml (R2 = 0.96, P < 0.0001)
and 1.0 ng/ml (R2 = 0.91, P < 0.0001), respectively. For plasma
with concentration of 1.0 ng/ml, the CV values of duplex real-time
PCR assay, real-time PCR with external standards and Picogreen
assay were 53.0%, 60.3% and 96.7%, respectively. In the
recovery test, there was a higher correlation of the plasma DNA
concentrations determined by the duplex real-time PCR assay
with the input DNA levels (R2 = 0.97, P < 0.0001) than those of the
real-time PCR with external standards (R2 = 0.85, P < 0.0001) and
PicoGreen assay (R2 = 0.92, P < 0.0001). Although remarkable
differences in plasma DNA concentrations determined by the realtime PCR with external standards and PicoGreen assay existed in
samples purified by the three different protocols (P = 0.0041 and
0.0000), there was no statistically significant difference in plasma
DNA concentrations among the methods by using our duplex realtime PCR assay (P = 0.5821). The median plasma DNA
concentration of females (16.9 ng/ml) was significantly lower than
males (22.6 ng/ml; Mann-Whitney two-sample rank sum test, P <
0.0001). Within the 95% confidence interval, the normal reference
interval of the plasma DNA concentration was 0~50 ng/ml for
males and 0~40 ng/ml for females. CONCLUSIONS: This newly
developed plasma DNA quantification method with the power of
eliminating variables introduced during plasma sample processing
allows more sensitive, repetable and accurate quantitative
measurement, and has a very promising future in clinical
application for diagnosis and disease monitoring.
Autocrine and paracrine role of leptin and
thrombopietin in controlling ovarian function in cow
Mihir Sarkar1,2, S Schilffarth1, D Schams1, HHD Meyer1, M W
Pfaffl1, S Ulbrich1, B Berisha1,3
1
Technical University Munich, Germany; 2Physiology &
Climatology, Indian Veterinary Research Institute, Izatnagar,
Bareilly, Uttar Pradesh, India; 3Faculty of Agriculture and
Veterinary, University of Prishtina, Prishtine, Kosovo
Leptin, the hormonal product of the obese (ob) gene, circulates in
the blood at levels paralleling those of fat reserves and regulates
satiety. In cattle, leptin has also been implicated in the control of
ovarian function, but its local production in the ovary and role in
the control of ovarian function in autocrine/paracrine manner is
unknown. Similarly, Thrombopoietin (TPO) is known to be involved
in mega-karyocytopoiesis, but its role in the control of ovarian
function is unknown in cattle. The aims of this study were to
demonstrate the expression of Leptin, its receptor (Ob-R), TPO
and its receptor (c-MPL) in detail in bovine corpus luteum (CL)
obtained from different stages of the oestrous cycle and during
pregnancy – and to demonstrate that leptin/Ob-R and TPO/ c-MPL
systems are expressed clearly in bovine follicles. Real-time RTPCR (qPCR) and ELISA were applied to investigate mRNA
expression of examined factors and leptin & TPO protein,
respectively. In general, we demonstrated leptin and its receptor
transcripts and leptin protein are consistent with in vivo
luteinisation of bovine CL and decline coincidental with luteal
regression. The highest co-expression of leptin/Ob-R system was
observed in TI and GC of the smallest follicles with E2
concentration <0.5 ng/ml followed by significant down regulation in
growing follicles with the increase of follicular size and E2 content
in the follicular fluid. Furthermore, expression of the leptin/Ob-R
system does not show any significant variation in the CL
throughout pregnancy. In this investigation, increases in the
concentrations of TPO protein and the mRNA expression of TPO
and c-MPL were noticed during both early luteal stage and late
luteal stage of the oestrous cycle. Furthermore, the expression of
TPO/ c-MPL system does not show any significant regulation in
the CL throughout pregnancy. Highest co-expression of TPO/ cMPL system in both theca interna (TI) and granulosa cells (GC) in
small follicles (<10 mm in diameter) was observed in this study
that may suggest the possible role of TPO/ c-MPL system in
proliferation of TI and GC cells. To conclude, our results are the
first to demonstrate the possible involvement of locally produced
leptin/Ob-R and TPO/ c-MPL system in the bovine ovary,
suggesting possible involvement of leptin/Ob-R system in the
function and/or development of the CL and growth of small follicles
in an autocrine/ paracrine fashion and of TPO/ c-MPL system as a
‘physiological filter’ in bovine ovary where they may promote cell
selection by inducing proliferation of viable cells and scavenging
non-viable cells and thereby may play an important role in
modulation of ovarian function.
ULTRA-RAPID REAL-TIME PCR for the detection of
viral diseases in honeybee
ByoungSu Yoon, MiSun Yoo, JiNa No, Van Phu Nguyen
Kyonggi University, Korea, South (Republic of)
Viral diseases in honeybee are very difficult to be identified. A
novel micro PCR-based detection method, termed ultra-rapid realtime PCR (URRT-PCR), was applied to the development of a
rapid detection for pathogenic virus in honeybee quantitatively,
such as IAPV, BQCV. In the URRT PCR assays showed high
sensitivities and accuracies in the both standard assays and
infected bees. In the application of URRT PCR detection from
infected honeybee, the detection time was only within 8 min,
including melting temperature analysis, performing 35 cycled
PCR. This novel detection method is one of the most rapid realtime PCR-based diagnostic tools and is expected to be applied to
the development of a rapid detection for various pathogens.
Development of quantitative triplex real time PCR for
the simultaneous detection of Mycobacterium avium
subsp. avium and M. a. subsp. hominissuis
Iva Slana, Maria Kaevska, Petr Kralik, Alice Horvathova, Ivo
Pavlik
Mycobacterium avium subsp. avium (MAA) and M. a. subsp.
hominissuis (MAH) belong to the Mycobacterium avium complex
and are frequently associated with diseases in animals and
humans. In animals it causes tuberculous lesions in
parenchymatous organs. Infections with MAA and MAH in humans
are rather scarce, but when present they cause severe
complications that lead to the chronic stage or sometimes even to
the death of individual. The aim of this study was to develop a
system for rapid and accurate quantitative real time PCR (qPCR)
identification and quantification of MAA and MAH. The developed
triplex qPCR reaction was based on the simultaneous detection of
specific insertion sequences, IS901 and IS1245 and an internal
amplification control. The specificity and sensitivity of the qPCR
were determined as well as the limit of detection for both
pathogens isolated from tissue samples. In order to quantify both
pathogens as precise as possible, the recovery of MAA and MAH
cells after DNA isolation was established. To test the triplex qPCR
assay coupled with the DNA isolation, tissue samples from 22 per
os artificially infected pigs, of which ten were infected with MAA,
ten with MAH and two were present as a negative control group
were tested. From each animal, 21 different tissue samples as
well as blood were tested by microscopy, culture and qPCR. In
both groups of experimentally infected animals, the newly
developed triplex qPCR assay proved to be more specific and
sensitive in comparison with the other methods used. Contrary to
culture examination, triplex qPCR confirmed the infection in all
animals infected with MAA, and in eight animals infected with
MAH. In conclusion, we developed a quick and sufficiently
sensitive triplex qPCR for MAA and MAH detection in tissue
samples that represents a suitable alternative to the culture.
This work was supported by the Ministry of Education, Youth and Sports of
the Czech Republic “AdmireVet” (CZ 1.05/2.1.00/01.0006 and No. ED
0006/01/01), the Ministry of Agriculture of the Czech Republic (Grant No.
MZe0002716202) and the EU grant PathogenCombat.
Rapid KRAS, EGFR, BRAF and PIK3CA Mutation
Analysis of Fine Needle Aspirates using allele-specific
qPCR
Ronald van Eijk, Jappe Licht, Hans Morreau, Tom van Wezel
Leiden University Medical Center, Netherlands, The
Fine needle aspiration based novel techniques for the diagnosis
and staging of diverse cancers have been incorporated into
cancer staging guidelines. The concordance rate of the mutation
analysis between these cytological aspirates and histological
samples obtained by surgical staging is often unknown. Therefore,
we studied the extent to which allele-specific quantitative real-time
PCR with hydrolysis probes could be reliably performed on fine
needle aspirates by comparing the results with histological
material from the same patient. Assays for the detection of seven
different KRAS, three PIK3CA and one BRAF variant together
with two different EGFR assays have been used on a series of
patients for whom cytological and histological material was
available. Raw data from the LC480 or CFX384 software were
imported into an in-house–created Microsoft Excel 2003
spreadsheet to define the mutation status. We demonstrated that
these standard molecular techniques can be accurately applied on
fine needle cytological aspirates.
Data Analysis: qPCR BioStatistics &
BioInformatics
Time:
Location:
Session Chair:
Wednesday, 30/03/2011: 9:00am - 12:00pm
Lecture hall 15
Michael W Pfaffl, Ales Tichopad
Interpretation requires context - making sense out of
gene lists and networks.
Philip Zimmermann
ETH Zürich, Switzerland
Many functional genomics and systems biology approaches result
in lists of genes with common properties. The interpretation of a
list of genes is crucial in understanding the results of such an
experiment. Typical approaches are to study gene enrichment of
pathway or GO categories, such as to find out which biological
processes are most affected in a given experiment.
Our work has focused on interpreting genes and gene lists by
bringing them into the context of a wide variety of experimental
conditions. For example, finding out in which tissues, at what time,
and in response to which factors a gene is expressed helps
understanding the function of this gene and its potential role in
one's own experiment. To achieve this, we manually curated,
annotated, quality controlled and normalized more than 55'000
expression microarrays from thousands of public experiments, and
created user-friendly tools to visualize gene expression across
these contexts. Several such meta-analysis tools are freely
available at www.genevestigator.com
Stratified error in the qPCR assays from the statistical
point of view
Ales Tichopad
Academy of Science of Czech Republic, Czech Republic
Statistical error affects quantitative PCR at various steps of
sample processing as well as during preparation of a standard
material. Quantification efficiency is most commonly evaluated
from the slope of standard curve serial concentrations projected
against the cycle of quantification. The efficiency is considered
contributing information to accurately calculate gene expression
relative to reference sample and/or reference transcript. Yet, what
is the reproducibility of the amplification efficiency estimation in an
experimental setup commonly used in labs? Experiment design
with repeated preparation of the standard curve was used to
estimate the uncertainty associated with estimation of
amplification efficiency and its impact on the gene regulation factor
calculated. We show that the additive error component due to
amplification efficiency estimation may deter the overall gene
regulation factor significantly. In addition, we show how various
sample processing steps affect the uncertainty of the regulation
factor.
Primer3: Improvements for the design of qPCR
primers
Andreas Untergasser1, Ioana Cutcutache2, Steve Rozen2
1
University of Heidelberg, Germany; 2Duke-NUS Graduate
Medical School, Singapore
For over one decade Primer3 assisted scientists in the process of
primer selection. The popularity of the Primer3 software in the
scientific community is indicated by over 5000 citations of the
initial publication. Several software tools employ Primer3 for the
primary selection of primers and reevaluate the output according
to their special needs, like the tool primer-blast at NCBI. Primerblast uses Primer3 for the selection of primers and evaluates their
specificity by blasting them against one of the NCBI nucleotide
databases. We think the success of Primer3 is based on the
accuracy of primer selection, the fast and stable implementation,
the flexibility of the primer selection process and the open source
license which allows the free use, modification and distribution of
Primer3. Users of Primer3 fall into three groups: bioinformaticians,
who embed Primer3 within their web servers, bioinformatics
scripts or pipelines, expert users who select large numbers of
primers and need tight control over the selection process, and
occasional users who simply want a convenient way to design a
few primer pairs. Bioinformaticians usually rely on the Primer3
command-line interface and run Primer3 on a local machine
(download at sourceforge.net/projects/primer3/). Expert or
occasional users usually rely on the Primer3 web interface,
Primer3plus (www.primer3plus.com/cgi-bin/dev/primer3plus.cgi).
The functionality of Primer3 has expanded in the last decade
considerably since it was first released. Primer3 now offers a
cleaner interface on the command line, improved primer selection
algorithms and up to date melting temperature calculations as well
as thermodynamic secondary structure calculations. The primary
function of Primer3 is the selection of primers for the amplification
a given sequence. The latest version can also pick lists of primers
which are not matched into primer pairs, primers required for
Sanger-sequencing, cloning primers, and can reevaluate userspecified primer pairs. The old algorithm had the tendency to over
represent very good primers in the result list and to favor certain
regions. To overcome this limitation, a region around a selected
primer can be defined, in which no second primer is allowed to
bind and thereby creating a list of truly unique primer pairs which
are well distributed over the sequence. In qPCR applications it is
desired to discriminate DNA derived from mRNA from genomic
DNA by using primers spanning an exon-exon junction. Primer3
can be provided with a list of junctions and will ensure that either
the forward or the reverse primer span one of these junctions.
Primer3 is a complex tool, that allows control of the selection
process with many parameters. Primer3 now has the option to
save settings for these parameters in a file, enabling expert users
to easily provide optimal parameter settings to less experienced
users. We are confident that the new modifications position
Primer3 to be a valuable for the coming decade.
Is normalisation of raw data necessary? Truly
Automated Analysis of qPCR Data Using the
AzurePCR Method
David Kennard, Ze'ev Russak
Azure PCR Limited, United Kingdom
Taking on molecular diagnostics industry challenges of interpreting
cycle threshold, creating normalised curves and using 'pretty
pictures' to facilitate manual interpretation of results, Azure PCR
will demonstrate a novel and unique externally validated method
for automated analysis of real-time (qPCR) data, including
classification and quantification. Unlike existing software included
within PCR cyclers, the AzurePCR (TM) automated analysis
method is assumption-free and does not require setting of user
parameters and, thus delivering time savings for both the
researcher and the clinician. This automated analysis method
demonstrates close to 100% and even 100% accuracy of
detection, as confirmed by recent validation studies, including for
data sets with high levels of background noise. We will address
current industry failings by comparing our results against those of
analysis software packages bundled with popular PCR cyclers.
This will be done using recent empirical validations of the
AzurePCR automated analysis method conducted with well-known
research hospitals and commercial laboratories.
Amplification efficiency as a function of primer and
cDNA concentration
Jan M Ruijter1, Quinn D Gunst1, Peter Lorenz2, Maurice JB
vandenHoff1
1
Academic Medical Center, Amsterdam, Netherlands, The;
2
University of Rostock, Rostock, Germany
Quantitative PCR data analysis results in strongly biased results
when the PCR efficiency is assumed to be 2 (100% efficient) or
when PCR efficiencies of target and reference genes are assumed
to be equal. The amplification efficiency (E) of a single PCR
reaction can be derived from the slope of the log-linear part of
individual amplification curves. The target quantity of the
amplicon-of-interest can then be calculated as N0 = Nq / E ^ Cq
(in which Nq is the quantification threshold). It has been shown
that the average of the individual PCR efficiencies per amplicon
gives an accurate and precise estimate of the target quantity per
sample. However, the causes for the observation of variable PCR
efficiencies per amplicon remain largely obscure. Optimizing the
primer concentration in a Sybr Green assay shows that decrease
of the primer concentration results in a decreasing observed PCR
efficiency. When the target quantities are calculated for these
samples, using the mean PCR efficiency per primer concentration,
the resulting N0 values are mostly correct. So, for part of the
range of primer concentrations the relation between observed
PCR efficiency and observed Cq value, Cq = {Log(Nq) - Log(N0)} /
Log(E), guarantees that the observed target quantity remains
unbiased. This also shows that in this range of primer
concentrations the PCR efficiency is a constant value from cycle 1
onwards. However, when the primer concentration is too low, the
PCR efficiency - Cq relation does not compensate for the resulting
low PCR efficiency and a significantly higher N0 value is
observed. A similar dependency of PCR efficiencies and Cq
values can be observed for the cDNA input concentration. This
shows that per qPCR assay the primer concentration should be
optimized for the range of target quantities that is expected to be
present in the samples. Moreover, samples with deviating
individual PCR efficiencies should be scrutinized when they result
in very high target quantities and, therefore, may have been
subjected to relatively low primer concentrations.
Statistical issues to consider when using qPCR to
validate microarray or RNA-Seq results.
Zaneta Anne Park
AgResearch, New Zealand
This presentation discusses statistical issues to consider when
running a qPCR experiment. These include the importance of
replication and randomization of treatments on each plate, and
using an experimental design to allocate samples so that the
treatments are balanced across runs. The importance of these
factors is emphasized by the presentation of data from a
uniformity trial. We also discuss statistical issues to consider when
creating standard curves. Additionally we present a novel analysis
method in which a new variable which incorporates the efficiency
of each gene is first calculated, and then the data analysed using
an Analysis of Variance. We have found this method easy to
implement and also useful for identifying outliers. A comparison of
the results using this method and those from REST1 are
presented.
1
Pfaffl, M et al. 2002. Relative expression software tool (REST©) for groupwise comparison and statistical analysis of relative expression results in
real-time PCR. Nucleic Acids Research, Vol 30, no. 9
Evaluation and applicability of advanced/exotic qPCR
quantification strategies and their implementation
University Hospital Hamburg-Eppendorf, Germany
Over its now three years of existence and development, the 'qpcR'
package for the R statistical environment has matured into a
quantitative real-time PCR (qPCR) data analysis software that
houses nearly 70 functions and example datasets assisting in
qPCR analysis. The versatile R statistical language makes it
relatively effortless to implement published algorithms or new
ideas in a short time, and test these new algortihms by the 20
included datasets derived from many different PCR platforms. The
package includes in its current version (1.3-4) many ‘replications’
of widely-used methods (i.e. window-of-linearity, REST approach,
exponential fitting, sigmoidal fitting, threshold cycle estimation by
second derivative maximum, Cy0, comparative quantitation, ratio
calculation in a batch format, etc). All of these methods have been
compared with the original versions, as to ensure result
compatibility/identity. Many implemented functions have also been
officially approved by the original authors. The talk will focus on
somewhat exotic implementations of qPCR quantitation that are
relatively new. While some of them are already published (but not
necessarily available in a software), we will introduce methods that
are still under development or amenable to public discussion in
respect to their general applicability within the qPCR community.
The methods that will be presented with short examples are:
1) Monte-Carlo simulation of error propagation (short).
2) Bootstrapping qPCR fits.
3) The development of a tilted threshold, which gives significantly
better calibration values from high dilution line by an iterative
process.
4) A six-parameter sigmoidal model which models the baseline
slope accurately.
5) The MAK2/MAK3 mechanistic models from Boggy et al.
6) The maxRatio method from Shain et al.
7) Several methods for automatic tagging of unsuccessful runs.
8) Using the PRESS statistic for goodness-of-fit.
9) The exceptional speed of R when analyzing high-throughput
data. We show this this on a dataset with 9000 runs (raw
fluorescence data), whose import, model fitting, quality control
(failed runs detection), threshold cycle and efficiency calculation
takes about 10 minutes.
10) The importance of weighting when fitting nonlinear models to
qPCR data.
Some these methods will be presented as short animations, to
clarify things. We will also show the feasibility of the methods by
testing them on a cohort of datasets.
High-resolution melting data error evaluation between
runs.
Maksim Bratchikov, Mykolas Mauricas
Centre for Innovative Medicine, State Research Institute, Vilnius,
Lithuania
High-resolution melting (HRM) based method for the bacterial
(Salmonella spp. as a target) genome typing was applied. The
targets for this typing were chosen from different variable
sequences. All raw data was derived on Rotor-Gene 65H0-100.
Slight but essential HRM variations between runs were found for
same samples tested. Therefore, reference samples for this HRM
error evaluation between runs were included. The calculations we
suggested were applied for raw data error normalization between
runs and fluorescent normalization. We successfully normalized
melting errors between runs. Data of all samples tested can be
stored for the comparison with newly tested samples.
GENEX: data analysis lunch seminar
Time:
Location:
Session Chair:
Wednesday, 30/03/2011: 1:00pm - 2:00pm
Lecture hall 15
Mikael Kubista
Amin Forootan
Presentation of GenEx 5.3; the most user friendly and
powerful qPCR experimental design, data analysis and
data mining software yet released!
MIQE: state of the art & open discussion
Time:
Location:
Session Chair:
Wednesday, 30/03/2011: 2:00pm - 3:00pm
Lecture hall 15
Jo Vandesompele
Mikael Kubista
qPCR is today the preferred method for quantitative nucleic acid
analysis, and is being extensively used in routine as well as
research. Proper usage of qPCR requires careful planning of the
experiment including optimized design, followed by elimination of
systematic noise by proper handling of controls and references,
and mining of the data. This complex flow is made easy with
GenEx 5.3. With GenEx a small pilot study is easily planned that
will identify the sources of confounding variation to the data, aiding
the design of the main experiment and estimating the number of
subjects needed to secure sufficient statistical power. The
measured data including relevant experimental settings and
sample/assay information are then imported directly into GenEx
from Roche, Agilent, Eppendorf, Bio-Rad, Life Technologies,
Illumina and Fluidigm qPCR instruments eliminating the risk of
introducing error by manually entering experimental parameters,
where noise is reduced by proper normalization with standards
and references, and prepared for statistical analysis. Then, with
few clicks with the mouse on user friendly and clear panels,
groups are compared using statistical tests, or patterns revealed
based on powerful classification tools. All results are presented in
clear and intuitive high resolved graphs ready for publication.
http://genex.gene-quantification.info
RDML: state of the art & open discussion
Time:
Location:
Session Chair:
Wednesday, 30/03/2011: 3:30pm – 4:30pm
Lecture hall 15
Jan Hellemans
Andreas Untergasser
Abstracts - Poster presentations
Location: Foyer - lower level
Time:
Monday, 28/03/2011: 6:00pm - 9:00pm
Tuesday 29/03/2011: 12:00pm - 2:00pm
Wednesday 30/03/2011: 12:00pm - 2:00pm
All poster sessions will take place in parallel and will last from the
"Monday Evening Poster Session" till "Wednesday Lunch Poster
Session".
Please put on your poster on Sunday afternoon or Monday
morning (until noon), and remove it on Wednesday afternoon.
Please bring your own drawing pins. You can offer handouts at the
poster site.
methods can be designed to be used as DIVA (“differentiating
infected from vaccinated animals”) tests, discriminating vaccine
strains from field circulating viruses, which can be of great
significance when using live attenuated vaccines. Finally, PCR is
the starting point in molecular characterization and epidemiology
studies of circulating viruses, essential for tracing the origin and
evolution of a disease outbreak. More recently, portable PCR
machines and other simple low-cost PCR-based techniques are
being launched for on-site application, meaning the analysis of
samples can be performed in regional labs or even in the
farm/slaughter-house with basic equipment by non-specialized
personnel. Due to many viral animal diseases are prone to very
rapid spread and the identification of the involved virus is urgently
required, these new tools may become useful first line pen-side
tests in a short time
P002
Poster number
Poster session
P001 – P046
New qPCR Applications: Molecular
Diagnostics & Expression Profiling
New qPCR Applications: Method Optimisation
& Standardisation
qPCR Biostatistics & Bioinformatics
High Throughput qPCR & digital PCR Next
Genartion Sequencing
Single-cell qPCR qPCR from limited material
HRM & Methylation Studies
RNAi: microRNA – siRNA Applications
Late submission
P047 – P055
P056 – P061
P062 – P069
P070 – P075
P076 – P084
P085 – P090
P091
New qPCR Applications:
Molecular Diagnostics & Expression Profiling
P001
PCR applications for diagnosis in Animal Health
Jovita Fernandez-Pinero1, Montserrat Agüero2
1
Centro de Investigación en Sanidad Animal (CISA-INIA),
Valdeolmos, Spain; 2Laboratorio Central de Veterinaria (LCV,
MARM), Algete, Spain
Viral infectious diseases are a constant risk for livestock
production due to the economic and sanitary costs produced by
their entrance into a country. Once the causative agent is
identified, specific strict sanitary measures must be rapidly
implemented to avoid its spread, and to control and eradicate the
disease. Classical laboratory diagnosis of viral diseases includes
both direct and indirect tools, virus isolation being the “goldstandard” technique to confirm the presence of an etiological
agent. Around 15 years ago, the merging of the PCR completely
transformed the diagnostic pathway in the National Reference
Laboratories and soon became an essential tool. Many benefits
prompted its incorporation into routine lab work, high sensitivity,
superior to virus isolation, being of most value as allows the early
detection of pathogens even before the evidence of clinical signs
in infected animals. The high specificity, the analysis of a great
number of any kind of clinical samples within hours, and the
chance to test several pathogens in the same reaction (multiplex
assays), made PCR a basic tool for screening diagnostic step.
The introduction of the real-time PCR changed the workflow once
again in lab diagnosis, by reducing analysis time and
contamination risk, and increasing the sensitivity and applications
in diagnosis. Moreover, real-time PCR accompanied by the
appearance of robots in the market for sample preparation and
nucleic acid extraction steps, made real a completely automated
analysis procedure for a high throughput application. Now, PCR
has a wide range of main applications in Animal Health. It is
extensively used in surveillance, control and eradication programs
of the major viral diseases affecting livestock at national and
international levels. PCR is a valuable tool in the prevention of
new disease entrance, the control of biological products, or the
evaluation of vaccines efficacy. Generic PCR assays can be
developed for detection of new viruses within a genus/family or to
determine the subtype/serotype of the identified virus. Also, PCR
A Comparative qPCR Analysis of Apoptotic Genes in
Milk Somatic Cells of Cows with history of
Inconsistent Lactations
Nishant Varshney1, Surender Singh1, Manu Jamwal1,
Sudarshan Kumar1, Dhruba Malakar1, Jai Kumar Kaushik1,
Sunita Grover1, B S Prakash1, Bishnu P Mishra2, Tapas
Mukhopadhyay3, Ajay K Dang1, Ashok K Mohanty1
1
National Dairy Research Institute, Karnal, India; 2National Bureau
of Animal Genetic Resources, Karnal, India; 3National Centre for
Human Genome Studies and Research, Punjab University,
Chandigarh, India
Milk yield has been a major selection criterion for genetic
improvement in livestock. The milk producing ability of farm
animals differ significantly, which may be due to the expression of
different various signaling proteins, transcription factors, cell
survival and death factors in the lactating mammary gland. It has
been observed that indigenous cows undergo self-induced dry
phase earlier than their average lactation period. A change in
mammary cells number is a principal cause of declining
production. The rate of decline in milk yield with stage of lactation
is strongly influenced by the rate of apoprosis. In the present
study, we have analyzed the differential expression of various
apoptotic genes such as BCL2, BAD, BAX, STAT3, STAT5,
CASPASE3 and CASPASE9 in indigenous cows (Sahiwal breed)
between normal lactating vs. short lactating cows. We have used
the milk somatic cells to ascertain our hypothesis that elevated
expression of apoptotic genes may lead to short lactation in
lactating cows. Short lactating cows taken for the experiment had
a lactation length of 100-150 days and the milk yield decreased
substantially after this period. Five cows (Sahiwal cows) of 3rd
parity each and having history of short lactation and normal
lactation length were selected for the experiments. RNA was
isolated from milk somatic cells at different stages of lactation
such as early, peak, mid and late lactation stages. For
normalization of qPCR data, two housekeeping genes such as
PPP1R11 and β actin were taken as reference genes. To
understand the relative pattern of expression of the selected
genes, expression profile at peak lactation was taken as the
calibrator. After normalization, we observed that all the candidate
genes in short lactating cows exhibited elevated expression in
comparision to normal lactating cows. Expression level of proapoptotic factors such as BAD, BAX, STAT3, Caspase 3, Caspase
9 showed fold increase of 18.75, 22.28, 5.8, 5.6 and 4.6 times
respectively during early lactation and 16.54, 32.8, 5.1, 1.7 and
2.6 times respectively during mid lactation in short lactating cows
in comparision to normal lactating cows. Similar trend was
observed during late lactation. The level of pro-apoptotic Bcl2
exhibited 5.4 fold increase during early lactation in short lactating
cows and was subsequently unchanged during mid lactation and
decreased during late lactation. The level of STAT5 exhibited a
higher expression level of 4.6 to 7.1 fold in short lactating cows. A
high degree of up-regulation of approximately 17, 32, 5, 2 and 3
fold respectively of pro-apoptotic molecules BAD, BAX, STAT3,
CASPASE3 and CASPASE9 was observed in short lactating
animals at the time of abrupt decline in milk yield mimicking the
initiation of accelerated apoptosis. Higher expression of key
apoptotic factors during early lactation in short lactating animals
indicated early initiation of apoptosis leading to untimely cessation
of lactation.
P003
Constitutive expression of HSPA-1A gene in two
different sources of buffalo (Bubalus bubalis) embryos
Nath Amar, Saikumar G., Gade Nitin Eknath, Taru Sharma G.
Indian Vetrinary Research Institute, India
Among heat shock proteins (HSPs), HSP70 is a most abundant
and highly conserved protein which serves as molecular
chaperone and provides thermal tolerance. In this study, we
determined the transcript levels of stress-responsive, heat shock
protein 70.1 (HSPA1A) gene quantitatively by real-time RT-PCR
(RT-qPCR) in preimplantation developmental stages of in vitro
produced (IVP) and in vivo derived (IVD) buffalo embryos. The
results of RT-qPCR revealed that the transcript for HSPA1A
mRNA is constitutively expressed in oocytes, the preimplantation
developmental stages; 2-4 cell, 8-16 cell, morula and day 7
blastocyst of IVD embryos along with their counterpart IVP
embryos. Marked differences in the mRNA level was observed at
2-4 cell, 8-16 cell, morula and day 7 blastocyst stages between
the two embryo sources (IVD and IVP). Amongst IVP buffalo
embryos day 7 blastocyst (5764±0.29) had lower HSPA1A
transcript copies as compared to 2-4 cell (9278 ±0.12), 8-16 cell
(10849±0.14), and morula (9154±0.23). Although similar trend was
observed for IVD embryo stages but there was marked
decreament in transcript level; 2-4 cell (7865±0.34), 8-16 cell
(10023±0.26), morula (8014±0.18) and day 7 blastocyst
(4912±0.36) as compared to IVP embryos. It is concluded that
HSPA1A is synthesized and expressed in stage specific manner
throughout preimplantation development of buffalo embryos
derived in-vivo and in-vitro. It supports the hypothesis that
HSPA1A is synthesized to overcome cellular stress during in vitro
culture environment and IVP embryos express high levels of
HSPA1A compared to IVD embryos.
P004
Applicability of qPCR to ensure the quality of milk and
dairy products
C LEMAITRE, E LABBE, T RIBEIRO, A CHANGO, A ABDEL
NOUR
LaSalle Beauvais, France
In food processing industry, quality is an unavoidable element,
much for consumers’ health as for organoleptic quality of the
products. Milk processing is highly concerned because milk is
subjected to various contamination sources, before milking (soil,
food, water or excrement), during milking (milking people) and
after milking (storage, transport or processing). As qPCR is
booming, we wondered if it was applicable to ensure the quality of
milk and dairy products. In this view, we did a systematic review of
articles and reference works published since 1999 dealing with
these problems. We identified several pathogenic and spoiling
microorganisms such as Staphylococcus aureus, Escherichia coli,
Pseudomonas
fluorescens,
Mycobacterium
tuberculosis,
Aspergillus flavus. qPCR also allows the quantification of
adulteration e.g. cow’s milk in goat’s milk, and could be used to
detect Genetically Modified Organisms. It has been proven that
qPCR is a reliable method, that can be used on various stages of
the process in question (just after milking, before and after
transport, after industrial processing…).
P005
Expression study of developmental genes in in vitro
produced buffalo (Bubalus bubalis) embryos
generated by hand-made cloning and in vitro
fertilization
SUDEEPTA KUMAR PANDA, KARN PRATAP SINGH, AMAN
GEORGE, AMBIKA PRASSNA SAHA, RUCHI SHRAMA,
RADHEY SHAM MANIK, MANMOHAN SINGH CHAUHAN,
PRABHAT PALTA, SURESH KUMAR SINGLA
NATIONAL DAIRY RESAERCH INSTITUTE, India
High incidence of developmental abnormalities is the major
drawback of nuclear transfer technique, which might be due to
insufficient reprogramming of somatic cell nuclei in enucleated
oocytes. The aim of this proposed study was to examine and
compare the expression of developmental genes in buffalo
embryos produced by hand-made cloning (HMC) which was
compared with in vitro fertilization (IVF). Total RNA was extracted
using cell to cDNA II kit (Ambion, Austin, TX, USA) from pools of
immature and in vitro matured oocytes (IVM) (n=20 each) and
embryos at the morula and blastocyst stages (n=5 each) for both
IVF and HMC groups for performing RT-PCR. Each experiment
was repeated 5 times. The data was analysed using one way
ANOVA after arcsine transformation of percentage values. The
pro apoptotic Bax gene expression was found to be not
significantly (P<0.01) differed in between morula and blastocyst
stages of embryo produced by both IVF and HMC, and was
undetectable in IVM oocyte. In another case, anti apoptotic Bcl-xl
expression was not significantly differed (P<0.05) in all
developmental stages of embryo produced by IVF, whereas its
expression in morula was significantly higher (P<0.05) than all the
stages of embryos produced by HMC. IVF morula has significantly
lower (P<0.05) Bcl-xl expression as compared to HMC morula.
These results indicated that All the stages of embryo produced by
HMC had significantly higher (P<0.05) Bcl-xl expression as
compared to Bax. The expression of Glut-1 was significantly
higher (P<0.05) in immature oocytes as compared to IVMoocytes,
morula and blastocyst stage in both IVF and HMC. Glut-1
expression was significantly higher (P<0.05) in blastocyst stage
produced by HMC in contrast to IVMoocytes, and blastocysts
produced by IVF. Whereas no significant difference was observed
in its expression during morula stage embryos produced by both
IVF and HMC. The HSP 70.1 was found to be significantly higher
(P<0.05) in immature oocytes compared to that in IVM oocytes
which in turn, was significantly higher (P<0.05) than that in
morulae and blastocysts produced by IVF as well as by HMC.
HSP 70.1 was significantly higher (P<0.05) in morula and
blastocyst stage embryos produced by IVF as compared to HMC
embryos, which was also undetectable in HMC blastocyst. These
results indicate that all developmental genes may be down
regulated during embryonic development from immature oocyte to
blastocyst stage embryos in HMC as compared to IVF. The
establishment of diagnostic techniques to determine cloned
embryos with the potential to develop into normal calves before
embryo transfer is crucial for the application of somatic cell NT to
animal husbandry.
P006
Validation of RT-qPCR measurements in primary
bovine mammary gland epithelial cells (pbMEC)
Diana Sorg, Anne Potzel, Heinrich H. D. Meyer, Enrique
Viturro, Heike Kliem
Technische Universität München, Germany
The inner surface of the bovine mammary gland is covered by a
layer of epithelial cells. These mammary gland epithelial cells
(MEC) synthesize the ingredients of the milk and secrete it into the
lumen. They also function as the first barrier to pathogens that
infect the udder. Cell lines and primary cultures of these cells are
often used for in vitro studies of inflammation and metabolism in
the udder. The common technique for obtaining these cells is
cultivating a piece of udder tissue after biopsy or necropsy.
Another technique is extracting primary bovine MEC (pbMEC)
from fresh milk. Little is known, however, about the changes in
gene expression of these cells in the course of cultivation. In this
study the aim was to measure the gene expression from pbMEC
over the course of the first three passages and to compare cells
taken from tissue (n=4) and from milk (n=3). In addition, the effect
of cryopreservation in liquid nitrogen on gene expression was
analysed. Therefore, 2 reference genes for normalisation (GAPDH
and Histon) and 8 target genes (Caspase 3, Cytokeratin 8,
HMGCR, IL1B, κ-Casein, RANTES, SREBP2 and TLR2) were
measured. None of the studied factors (cell origin, passage
number, freezing) had any significant effect on gene expression
levels (p >0.05). pbMEC extraction from milk is non-invasive and
repeatable. These are advantages in comparison to tissue
sampling. Our results suggest that these cells can be equally
taken from both origins regarding the effects on gene expression
in cell culture. Also they can be used during the first three
passages without significant alteration of the gene expression
pattern. Cryopreservation in liquid nitrogen is often necessary
preparing an in vitro experiment. This can also be done without
influencing gene expression of the pbMEC.
P007
P009
Heterogeneity within the pluripotent stem cell state is
associated with transient changes in gene expression
Standardisation of triplex real time RT-PCR for
detection and quantification of hepatitis E virus
Janice K Au-Young1, Jason Gioia1, Srividya Dadi1, David
Keys1, Paul J Gokhale2, Peter Andrews2
1
Life Technologies, United States of America; 2Centre for Stem
Cell Biology, University of Sheffield, UK
Petra Vasickova, Petr Kralik, Iva Slana, Ivo Pavlik
Insight into the molecular, surface marker and epigenetic profile
that makes up the pluripotent state of embryonic stem cells from
both the laboratory mouse and humans has been gained recently.
Previous studies showed that human embryonic stem cell (hESC)
cultures are made up of heterogeneous populations that are
positive or negative for the cell surface marker SSEA3. SSEA3
positive cells have a higher percentage of colony formation and
higher levels of pluripotency markers than SSEA3 negative cells.
Moreover, different hESC isolates display different propensities for
differentiation. One obvious source of variation between cell lines
is epigenetic differences caused by establishment in culture and
prolonged passage. However a more subtle ‘intrinsic’ form of
heterogeneity may exist, wherein a given cell compartment
defined by a surface marker, has differences in gene expression
and thus differentiation potential. Our studies of hESCs at the
single cell level provide further support for a model in which
undifferentiated cells can oscillate between cell states within a
more general stem cell compartment which is associated with
transient changes in gene expression. Upon culture adaptation,
a lower frequency of cells express lineage specific transcripts
compared to normal hESC cultures. Moreover, functional testing
for cells undergoing spontaneous differentiation showed that
adapted hES cell populations are less likely to have endodermal
markers
compared
to
normal
cultures.
P008
Impact of long-term expansion on mesenchymal
stromal cell differentiation potential
Andrea Hecker, Irena Brinkmann, Karen Bieback
Institute of Transfusion Medicine and Immunology, Medical
Faculty Mannheim
Mesenchymal stromal cells (MSCs) are promising candidates for
novel cell therapeutic applications. To obtain a clinically relevant
dose, expansion seems to be required. Long-term culture,
however, can induce spontaneous transformation. Thus we
investigated whether long-term expansion affects MSCs qualities
and analysed MSC differentiation capacities by histochemistry,
colorimetric assays and reverse transcription quantitative PCR
(RT-qPCR). For an exact comparison of mRNA transcription
conform to MIQE guidelines, RT-qPCR includes internal standards
such as reference genes (RG) to normalise mRNA levels between
different samples. Classical RG however are deregulated by
differentiation processes and thus may lead to unreliable results.
For accurate RT-qPCR expression profiling of differentiation
markers, we used geNorm software to determine the most stable
RG from a set of five reference genes. Each three osteogenic and
adipogenic induced and corresponding noninduced MSCs
samples were measured by qPCR and the data analysed by the
geNorm software. For each primer pair the PCR efficiency was
calculated by a standard curve derived of a pool of differentiated
and undifferentiated MSCs. Interestingly, different RG sets were
identified for adipogenic and osteogenic differentiation pathways:
GAPDH, B2M and SFRS4 were found to be the most stable genes
for adipogenic and TBP, SFRS4 and B2M for the osteogenic gene
expression. From this set, a normalization factor (NF) was
calculated and used to determine relative gene expression of
osteogenic and adipogenic markers of long-term cultivated MSCs.
Our results revealed that MSC undergo replicative aging which
rapidly affects adipogenic differentiation potential. Osteogenic
potential appeared less affected by the culture age of MSCs.
Moreover our data underline the necessity to carefully assess
appropriate and valid normalisation controls for RT-qPCR.
Hepatitis E virus (HEV) is the main causative agent of hepatitis
non-A, non-B in humans worldwide. The virus is non-enveloped
and its genome contains single-stranded, positive-sense RNA 7.2
kb in length. According to sequence comparisons and
phylogenetic analysis HEV isolates segregate at least into four
major genotypes and 24 subtypes. Genotypes I and II are
restricted to humans and often associated to epidemics in
developing countries with poor sanitation and hygiene. To the
contrary, genotypes III and IV are responsible for sporadic cases
of hepatitis E in both developing and industrialized countries.
These genotypes were found in samples of human and animal
origin and thus their zoonotic transmission has been considered.
Due to the lack of an efficient cell culture system, the most
common methods of HEV detection are techniques based on
PCR. HEV genome, as of all RNA viruses, is very polymorphic
and thus a single locus PCR fails to detect all possible HEV
genomic variants. Therefore the aim of this study was to
standardise a two-tube reverse transcription triplex quantitative
real time PCR (RT-qPCR) for detection and quantification of
hepatitis E virus (HEV) RNA combining amplification of two HEV
loci with an internal amplification control (IAC). Czech strain
CZswHEV1 (EU117408), which belongs to the most common
subtype detected in the Czech Republic, was used as a template
for RNA standards. These RNA standards as well as IAC were
prepared by in vitro transcription and subsequently used for
standardisation of assay. The limit of detection was experimentally
determined as 10 copies/µl of the RNA standard for both
amplification targets. The presented triplex RT-qPCR assay can
detect at least 10 copies of the HEV genome/µl of isolated RNA
and allows quantification based on RNA standards. Usage of RNA
IAC facilitates monitoring of false negative results. Based on these
data, the assay represents significant improvement of HEV RNA
detection.
This work was supported by the Ministry of Agriculture (No.
MZE0002716202), the Ministry of Education, Youth and Sports of Czech
Republic (No.OC08045 and CZ 1.05/2.1.00/01.0006 AdmireVet) and by EC
Grant Cost Action 929 ENVIRONET.
P010
Molecular basis for the impaired adipogenic
differentiation potential of cord-blood-derived
mesenchymal stromal cells
Irena Brinkmann, Andrea Hecker, Marianna Karagianni, Karen
Bieback
Institute of Transfusion Medicine and Immunology, Germany
Mesenchymal stromal cells (MSCs) have received considerable
attention for their potential role in cell-based regenerative therapy.
For their clinical application, a better understanding of the
behaviour of the cells, e.g. MSCs from different tissue sources,
including differentiation, proliferation and migration and the
disparities among them is required. MSCs have the ability to
differentiate into different cell types such as bone, cartilage and
fat. Cord blood (CB) derived MSCs, in contrast to the MSCs
isolated from lipoaspirate (LA) and bone marrow (BM), differ with
respect to low or absent adipogenic but higher osteogenic
differentiation potential in vitro. Preadipocyte factor-1 (Pref-1,
DLK-1) has been shown via reverse transcription quantitative PCR
(RT-qPCR) to be contrarily expressed in CB MSCs in comparison
to LA and BM MSCs during adipogenic differentiation. CB plasma,
containing high levels of Pref-1, has shown to inhibit adipocyte
differentiation in LA MSCs, indicating a potential role of Pref-1 in
inhibition of adipogenesis. Accordingly, we postulated that Pref-1
knockdown via siRNA should induce an adipogenic phenotype in
CB MSCs. Unexpectedly; it was not possible to detect adipogenic
differentiation after 21 days of Pref-1 knockdown, neither on RNA
level nor as lipid vacuoles. Further studies should ascertain
whether Pref-1 protein is still abundant after 21 days of siRNA
treatment to inhibit the adipogenic differentiation in CB MSCs. In
addition a screening for Pref-1 interaction partners will be
performed to define whether Pref-1 requires specific proteins for
their inhibitory role in adipogenesis. Our studies have identified
Pref-1 as one candidate to be responsible for the impaired
adipogenic differentiation potential of CB MSCs. It needs to be
investigated if Pref-1 competitively balances the differentiation into
the adipogenic and osteogenic lineage. Thereby a high Pref-1
expression, that yields a lower or absent adipogenic
differentiation, may consequence a higher osteogenic
differentiation potential.
P013
P011
Quantification of genetically modified organisms (GMO) in food or
feed samples in routine laboratories is primarily done by relating
the copy number for a GMO-specific inserted gene with the copy
number of a species-specific reference gene. The ratio gives a
copy-based percentage which is then converted into a weightbased percentage of GMO contents specified in official results.
The copy numbers for GMO and reference gene are determined
by two separate quantitative real-time PCR assays with standard
curves using TaqMan chemistry [1]. Measurement uncertainty
(MU) is an essential characteristic of experimental results. There
are many comprehensive guides for estimation of MU available
[e.g. 2, 3]. Unfortunately, many of these guidelines have a rather
chemistry background and may not therefore be reliably applied to
real-time PCR measurements. Additionally, few publications exist
that deal especially with MU in GMO analysis [e.g. 4, 5, 6]. We
combined from these - often quite theoretical guidelines - a
practical approach for appraisalof MU. Uncertainties related to
precision, recovery, inhomogenity, and reference materials were
estimated and combined in an expanded MU with a confidence
level of 95 %. As quantification becomes more inaccurate with
decreasing GMO contents, we calculated separate uncertainties
for the relevant (legal) thresholds of 0.1 %, and 0.9 % GMO
content. Quality assurance (QA) requires the control of the
reaction parameters that form the basis of the measurements. We
developed a set of control charts for visualising the quality control
data of real-time PCR testing. Spreadsheet software-based
measurement analysis is combined with semi-automatic
documentation for QA purposes. The presented approaches have
become valuable tools in maintaining the high standards at the
Bavarian Health and Food Safety Authority (LGL), assured by
accreditation according to DIN EN ISO /IEC 17025:2005.
qPCR assay for detection of human faecal
contamination in food samples
Lotte Bjerrum1, Anna Charlotte Schultz2, Dorthe Lau
Baggesen2, Anna Krestine Nørgaard1, Aaron Marc Saunders3
1
Danish Technological Institute, Denmark; 2National Food
Institute, Technical University of Denmark; 3Aalborg University,
Denmark
Food and drinking water serves frequently as vehicles for
transmission of human enteric viruses like noroviruses.
Contamination of produce can take place during production by use
of sewage polluted water or inefficient hygienic norms. Currently,
there are no routine testing for viruses in foods and water due to
the lack of validated methods and the methods used in research
laboratories are still too labour-intensive and expensive to be
incorporated in the quality control of most food industries. In this
project we developed at molecular indicator tool to determine the
presence of human faecal pollution in relevant food sources and
growth environments. A qPCR assay that detects a specific
indicator organism, Bacteroides dorei, unambiguously linked to
human faeces/wastewater was developed. As extraction of RNA
and DNA from mollusks, fruits and vegetables can be challenging,
effort was put into optimizing the procedures for extraction, so that
both RNA (norovirus) and DNA (the bacteria) was retrieved as
efficient as possible. The optimisations steps as well as qPCR
results of norovirus and B.dorei will be presented. The impact of
the method and examples of its use in mollusks, raspberries and
other contaminated foods will also be presented.
P012
qPCR method to calculate the copy number of IS1111
elements in Coxiella burnetii
Matthias Hanczaruk, Anke Stark, Dimitrios Frangoulidis
Bundeswehr Institute of Microbiology, Germany
Coxiella (C.) burnetii, the causative agent of Q fever, is deemed to
have the potential to be used as a biological weapon. Therefore,
investigating outbreaks to trace back the source of an infection is
of high importance in forensic microbiology. The insertion
sequence IS1111, coding for a transposase, is used as a target
region for diagnostic PCRs, since it is found in multiple copies in
C. burnetii genomes. In silico analyses of the 7 annotated genome
sequences demonstrated copy numbers ranging from 12 to 52.
Chromosomal rearrangements and DNA insertions/deletions
involving these transposable elements are responsible for
genomic plasticity, leading to diversity among isolates, and may
have influence on virulence. Till now, whole genome sequencing
has been the only reliable method to determine exact IS1111 copy
numbers. However, this method is laborious, time-consuming,
cost-intensive and certainly not suitable to test many C. burnetii
isolates. We established a quantitative duplex real-time PCR
assay based on Taqman-technology, allowing the calculation of
IS1111 copy numbers. To this end, a FAM-labelled probe targets
the multi-copy IS1111 gene, while the single-copy COM1 gene,
encoding for an outer membrane protein, is detected in the HEXchannel. We used the previously sequenced strains Nine Mile and
Henzerling, with 20 and 47 IS1111 elements, respectively, to
calibrate the assay. In dilution series, we calculated the efficiency
of the primer-probe-combinations and included the results in our
evaluation. We tested a selected panel of 100 C. burnetii isolates
and demonstrated that the number of IS1111 elements can vary
from 20 to more than 100 copies. It has been assumed that the
copy number of IS1111 elements can be influenced during
multiple cell culture or animal passages. With the method
presented here we could prove this thesis and also will have a
marker tool, easy to determine, to control inner herd or other
population related genomic variations of Coxiella burnetii strains.
Quality assurance in GMO analysis – A practical
approach for estimation of measurement uncertainty
and visualisation of quantitative real-time PCR data
Lars Gerdes, Wilhelm Dicke, Ulrich Busch, Sven Pecoraro
1. European Union Reference Laboratory for GM Food and Feed (EURLGMFF). http://gmo-crl.jrc.ec.europa.eu/.
2. ISO, GUM: Guide to the
expression of uncertainty in measurement, ISO, 1995. 3. ISO, ISO/TS
21748: Guidance for the use of repeatability, reproducibility and trueness
estimates in measurement uncertainty estimation, ISO, 2004. 1-30.
4.
Burns, M. and H. Valdivia, A procedural approach for the identification of
sources of uncertainty associated with GM quantification and real-time
quantitative PCR measurements. Eur Food Res Technol, 2007. 226: 7-18.
5. Trapmann, S., et al., Guidance document on measurement uncertainty
for GMO testing laboratories, JRC, 2007. 1-41. 6. Žel, J., et al., Calculation
of measurement uncertainty in quantitative analysis of genetically modified
organisms using intermediate precision--a practical approach. J AOAC Int,
2007. 90(2): 582-6.
P014
Quantitative Real Time PCR in Friedreich’s Ataxia:
implications for diagnosis and clinical trial design.
Giorgia Puorro1, Antonella Antenora1, Angela Marsili1,
Alessandra Denaro1, Raffaele Piro1, Pierpaolo Sorrentino1,
Chiara Pane1, Alessandra Tessa2, Sergio Cocozza3, Filippo
Santorelli2, Giuseppe De Michele1, Alessandro Filla1,
Francesco Saccà1
1
Dipartimento di Scienze Neurologiche, University Federico II,
Naples, Italy; 2Dipartimento Clinico di Neuroscienze e dell'Età
Evolutiva, IRCCS Stella Maris, Pisa, Italy; 3Dipartimento di
Biologia Cellulare e Molecolare, University Federico II, Naples,
Italy
Background: Friedreich’s ataxia (FRDA) is the most common
hereditary ataxia among caucasians. The molecular defect in
FRDA is the trinucleotide GAA expansion in the first intron of the
FXN gene, which encodes for frataxin. Aim of the study was to
screen a population of FRDA patients, carriers, and controls for
FXN mRNA levels, and to determine the utility of q-PCR as a
biomarker and diagnostic tool. Design/methods: We enrolled 24
patients with classic FRDA phenotype (cFA), 6 late onset FRDA
(LOFA), 5 compound heterozygotes for expansion and point
mutations (pFA; I154F, IVS4+3delA, R165P), 33 healthy
expansion carriers, and 30 healthy controls. DNA was genotyped
for GAA expansion. Total mRNA was extracted from PBMCs and
reverse transcribed using a one-step Master Mix. cDNA was
amplified in multiplex and standardized by quantification of HPRT1 as a reference gene. Relative expression was calculated using
healthy controls as a reference group using the efficiency
calibrated model. Analysis was performed with the improved
version of the relative expression software tool (REST 2009).
Continuous variables were analyzed using the one-way ANOVA.
Post-hoc analysis was performed with the Bonferroni multiple
comparison’s test. Diagnostic efficacy of FXN mRNA dosage was
assessed constructing ROC curves. Correlation analysis was
performed calculating Pearson’s coefficient. P values of less that
0.05 were considered statistically significant. Results: In cFA,
mRNA was profoundly reduced to 19.4% of controls (range 0.060.48, p<0.0001) whereas there was a less severe down-regulation
in pFA (52.7% of controls , p<0.0001), reulting different mRNA
relative expression levels between cFA and pFA (p<0.001). LOFA
patients and carriers showed a less severe down-regulation to
50.4% (range 0.35-0.85, p<0.0001) and 53.0% (range 0.11-1.21,
p<0.0001), respectively. Comparison of cFA vs controls, resulted
in 100% sensitivity and specificity for a cut-off frataxin mRNA
value of 0.6 as compared to controls (p<0.0001, area=1.00).
Comparison of pFA with controls resulted in a sensitivity of 100%
and specificity of 88% for a cut-off frataxin mRNA value of 0.75
(p<0.001, area=0.976). For mRNA correlation analysis, cFA and
LOFA were considered together as a single group. mRNA levels
correlated directly with age at onset (P<0.001, R2=0.3905) and
inversely with GAA1 and GAA2 (p<0.01, R2=0.2364; p<0.05,
R2=0.1750). We found an inverse correlation between mRNA
levels and GAA2 in carriers (p<0.0001, R2=0.5011). Conclusion:
We report the first explorative study on frataxin mRNA levels in
PBMCs from a cohort of FRDA patients, carriers and healthy
controls. FRDA patients showed reduced levels of FXN mRNA to
one-fith of control levels. Messanger RNA levels proved to be
diagnostic when comparing cFA to controls with 100% sensitivity
and specificity. In contrast, q-PCR is not able to differentiate pFA
and controls. Clinical trials should be designed to include FXN
mRNA levels as an endpoint.
P015
P016
Quantization of Rauscher-like MuLV in plasma from
BALB/cJ mice
Yehuda Stram1, Marisol Rubinstein1, Evgenia Lubashevsky1,
Sofa Savranski1, Gavriel Leitner1, Zeev Treinin2
1
Kimron Veterinary Institute, Israel; 2Israel Dairy Board
Recently it was reported by Ter-Grigorov , et al., 1997 that a new
virus arose in BALB/c females which were repeatedly mated to
C57BL/6 (B6) males, and then injected with fixed, activated B6
male spleen cells. The clinical symptoms of the infected mice, by
the new virus, resembled those of AIDS in man. To characterize
the virus, BALB/cJ mice were inoculated with virus-containing
plasma which was derived from mice 3 weeks post infection.
Biological and molecular analysis of the new virus revealed a
novel virus mixture of murine leukemia viruses (MuLVs), termed
Rauscher-like MuLV, that causes an increase in hematopoiesis
due to activation of pluripotent HSC. It could be demonstrated that
endogenous BALB/c mouse ecotropic and xenotropic MuLVs are
activated by these treatments. multiple pregnancies and allostimuli
appear to have provided the signals required for activation of and
recombination among endogenous viruses and could have
resulted in generation of the Rauscher-like MuLV mixture. To
better understand the phenomena we aimed to determine the
amount of each of the viruses in the mixture. Taqman technology
was employed for this purpose. To establish standards for each
virus measurements, fragments from each of the viruses carrying
the Taqman amplicon were amplified in a way that the forwarded
primer carried the T7 promoter sequences. Each of the amplified
fragments were transcribed using T7 RNA polymerase. Ten fold
dilution of the newly transcribed fragment were used to establish
standard curves. It could be shown that about 10 viral RNA
molecules per sample could be detected using this system. The
developed technique will be used to study the underlined
molecular processes that leads to the production of this unique
viral mixture and its role in aids like disease in the affected mice
Cutaneous Mycobacterium marinum infection in
humans detected by quantitative real time PCR
P017
Michal Slany1, Krystyna Kantorova1, Petr Jezek2, Monika
Bodnarova3, Ivo Pavlik1
1
Veterinary research institute, Czech Republic; 2County Hospital
Pribram, Czech Republic; 3Charles University Praque, First
Faculty of Medicine and General University Hospital, Czech
Republic
Development of a real-time quantitative multiplex
TaqMan-based RT-PCR assay for detection and
quantification of measles, mumps and rubella viruses
The low frequency of infection caused by non-tuberculous
mycobacteria (NTM), non specific symptoms for individual NTM,
requirements of specific cultivation approaches, very often are not
routinely used microbiological laboratories and the lack of specific
identification methods could detract from a correct diagnosis.
Mycobacterium marinum is occasionally associated with
cutaneous infections in humans so called “fish tank granuloma”.
Rapid, sensitive and specific method for the detection of
M. marinum in animal and human samples was set up in this
study. The duplex real-time PCR assays (erp) described here
provides a fast, sensitive and specific diagnosis of M. marinum, in
comparison to more time-consuming conventional PCR and DNA
hybridization methods. We developed a quick and sufficiently
sensitive system for the detection of M. marinum in tissue samples
with a clinical sensitivity of 5 × 102 CFU per 50 mg of tissue for erp
qPCR and with median of isolation yield 57.79%. Developed
qPCR system was successfully used for detection of M. marinum
infection in two humans. Interestingly, one of the patients was
previously misdiagnosed and treated with methylprednisolone for
period of 3 month, which resulted in a rare systemic spread of
M. marinum into the testis and epidimidis. Simultaneously, the
screening for M. marinum presence in both patients’ aquarium
environments revealed infected fish with M. marinum. The protocol
used enabled us to complete analysis of sample, including
controls, in approximately 6 hrs; including extraction and assay
time, and is suitable for implementation in routine laboratory
diagnostics. To our best knowledge this is the first report of
detection and quantification of M. marinum directly from infected
human tissue.
This work was supported by Grants Nos. MZE0002716202 and QH91240
from the Ministry of Agriculture and Grant “AdmireVet” No. CZ
1.05/2.1.00/01.0006-ED0006/01/01 from the Ministry of Education, Youth
and Sports of the Czech Republic.
Yulia Zabiyaka, Eugeny Faizuloev, Sergey Lobodanov,
Tatiana Konstantinovna Borisova
Mechnikov Research Institute of Vaccines and Sera, RAMS,
Russian Federation
Vaccine potency is a major property for the quality of live virus
vaccines. Potency assay of trivalent measles, mumps and rubella
(MMR) live virus attenuated vaccine is performed routinely by
plaque or conventional 50% cell culture infective dose (CCID50)
assays. However, these assays are time-consuming, laborintensive, typically highly variable and requiring the availability of
highly specific neutralizing antibodies in order to allow detection of
the infectivity of the individual components in multivalent vaccines.
In the present study, novel methods to estimate the potency of
MMR viruses in virus-containing bulks using real-time quantitative
TaqMan-based reverse-transcription PCR (qPCR-RT) have been
developed for each virus and the principal possibility of MMR virus
quantification using Multiplex assay format has been shown.
Specific primers and probes were designed and the qPCR-RT was
optimized by annealing temperature and MgCl2 and primer
concentration. The results determined by qPCR-RT method have
been compared in parallel with that of a conventional
CCID50 assay for MMR virus samples obtained daily during longterm cultivation and for the International Reference Reagents of
MMR vaccines (Live). For potency estimation by Multiplex qPCRRT and CCID50 assay laboratory variant of trivalent MMR vaccine
containing the L-16 measles, the L-3 mumps and the Wistar RA
27/3 rubella virus strains and commercial batches of MM divalent
(Microgen, Russia) and MMR vaccines (Priorix, GSK Biologicals,
Belgium) were used. The potency of the viruses determined by
qPCR-RT was estimated relative to the 10-fold dilutions of the
reference samples with titers 5,4, 5,2 and 6,3 lgCCID50/ml for
MMR viruses respectively and the corresponding trivalent sample.
To control tube-to-tube variations the Human Rhinovirus 16 as an
internal positive reference was used. It was found that the
difference between viral titer measured by qPCR-RT and the
corresponding infectious viral titer measured by CCID50 assay in
paired samples did not exceed 0,3 lgCCID50/ml for certain period
after inoculation (WHO allowed value) and the Pearson coefficient
between results obtained by two assays was close to 1, indicating
a significant correlation. For the reference samples of vaccines,
the discrepancy between the titers determined by qPCR-RT and
CCID50 assays was within 0,2 lgCCID50/ml, which is less than
WHO allowed value. The developed qPCR-RT method was shown
to have high efficiency, high sensitivity and high reproducibility.
This assay is virus-specific and serological neutralization can be
omitted, besides it was faster and less laborious compared to the
classical assay. While the traditional methods would still be
necessary to determine viral potency (in term of infectious virus),
the novel qPCR-RT was demonstrated to be allowed method for
rapid determination of optimal harvest time and bulk virus titers. It
also can be used for rapid detection of MMR viruses in clinical
specimens.
P018
Comparative study of direct sequencing and
TheraScreen assay for EGFR mutational analysis
LUZ MARTINEZ AVILES, JAVIER GIMENO, RAQUEL
LONGARON, ERICA TORRES, GEMMA NAVARRO, LARA
PIJUAN, SERGI SERRANO, BEATRIZ BELLOSILLO
HOSPITAL DEL MAR, Spain
BACKGROUND: Epidermal growth factor receptor tyrosine kinase
inhibitors are used in non-small cell lung cancer (NSCLC).
Patients carrying mutations in the EGFR gene have a better
response to this therapy than patients with wild type EGFR. AIM:
The aim of this study was to compare the analytical sensitivity of
two different procedures to detect EGFR mutations. METHODS:
We have determined the mutational status of the EGFR gene in
tumoral samples from 200 patients with NSCLC by two
methodologies: 1. PCR amplification of exons 18, 19, 20 and 21 of
the EGFR gene followed by direct sequencing using BigDye 3.1
and analysis in a genetic analyzer (Applied Biosystems) and 2.
TheraScreen (DxS) assays using a real time PCR system
(Applied Biosystems). RESULTS: Analysis of EGFR mutations in
200 biopsies of NSCLC patients by both techniques showed a
concordance of 97%. From the whole cohort, 169 patients were
negative for EGFR mutations whereas 31 patients presented
mutations (at least by one technique). From the group of EGFRpositive patients, in four cases the mutation was only detected by
the TheraScreen assay because the mutant allele burden was
very low. In two additional cases, the mutation was only detected
by direct sequencing. In the first one the patient carried the V769M
mutation, an alteration that is not included in the commercial kit. In
the second case the patient harboured the G719S mutation at
exon 18 (which is tested in the commercial kit), but also the E709A
mutation at the same exon that probably disrupted the binding of
the probe, so we obtained a false negative result with the
TheraScreen assay. CONCLUSIONS: Both direct sequencing and
TheraScreen assayshowed a good correlation although
TheraScreen assay has a greater sensitivity than direct
sequencing in samples with low mutant allele burden. The
TheraScreen assay may give some false negative results because
some infrequent mutations are not included in the test and
additional mutations near the ones tested in the kit may affect the
binding of the probes.
P019
Real-time PCR with SYBR Green and melting curve
analysis as a diagnostic screening tool for protozoan
oocysts
Laura F. Lalonde, Alvin A. Gajadhar
Canadian Food Inspection Agency, Canada
A simple screening method for the detection and identification of
protozoan oocysts is a necessary diagnostic tool for human,
animal, and food-borne disease outbreak investigations. Our
objective was to develop a real-time quantitative PCR (qPCR)
assay with melting curve analysis (MCA) to detect, differentiate,
and identify DNA from protozoan species of veterinary, zoonotic,
and food safety importance. We designed and employed a
universal coccidia primer cocktail consisting of seven
oligonucleotides to amplify 18S rDNA from Cryptosporidium
parvum, Toxoplasma gondii, Cyclospora cayetanensis, and
several species of Eimeria, Sarcocystis, and Isospora using qPCR
with SYBR Green detection. A standard curve was constructed
from DNA of serial dilutions of 105, 104, 103, 102, or 101T. gondii
oocysts to determine assay sensitivity. DNA from as few as 10 T.
gondii oocysts could be routinely detected. Melting temperatures
(Tm) were determined for each species based on MCA of multiple
replicates over several runs. Tm data analysis showed that C.
cayetanensis, C. parvum, Cryptosporidium muris, T. gondii,
Eimeria bovis, Eimeria acervulina, Isospora suis, and Sarcocystis
cruzi could each be identified by unique melting curves and
differentiated based on Tm. The qPCR MCA assay successfully
amplified the target gDNA of both C. parvum and C. cayetanensis
or S. cruzi and T. gondii when these species were together in a
mixed template. When used with suitable controls, this qPCR
MCA assay could be used to sensitively detect, reliably
differentiate, and rapidly identify DNA of protozoan oocysts in
faecal, food or clinical diagnostic samples using readily available
SYBR Green dye and without the need for a high-resolution MCA
qPCR instrument. Further validation of this simple qPCR MCA
assay using contaminated field samples and multiple isolates of
each species will support its application as a routine diagnostic
screening tool for parasitology.
P020
RNA isolation from high-sugar content freeze-dried
tomato (Solanum lycopersicum L.) fruits and qRT-PCR
analysis
Libert Brice TONFACK1,2, Emmanuel YOUMBI1, Benoit vander-Rest2, Alain LATCHE2, Jean-Claude PECH2
1
University of Yaoundé I, Cameroon; 2UMR990, Genomic et
Biotechnologie des Fruits INRA/INP-ENSAT, Toulouse, France
With minor modifications, we applied a previously reported RNA
isolation protocol that used phenol/chloroform/isoamylalcohol to
lyophilized (freeze-dried) fruits of tomato (Solanum lycopersicum
L.). When gene expression profiles are studied, plant material
must be preserved in its collected state. Fresh plant material
cannot feasibly be transferred at ultra-low temperatures from
natural habitats to the laboratory. We explored the use of
lyophilized tissue for RNA isolation from two varieties of tomato
fruits. High yields of RNA (~236 μg/g dry weight of fruit tissue)
were obtained, and the RNA was suitable for all molecular biology
methods tested, including quantitative reverse transcription Real
Time Polymerase Chain Reaction (qRT-PCR) analysis. We
demonstrated that RNA obtained from freeze-dried fruit tissue was
of good quality, undegraded, and useful for all molecular
downstream applications. This work opens an important ways to
improve and sustain researches in conditions of lack of modern
tools for better development.
P021
Seasonal quantification of Grapevine fanleaf virus by
one-step RT real-time PCR
Urska Cepin, Ion Gutiérrez-Aguirre, Maruša Pompe-Novak,
Kristina Gruden, Maja Ravnikar
National Institute of Biology, Slovenia
Grapevine fanleaf virus (GFLV) is a +ssRNA virus, which is the
causal agent of the fanleaf degeneration disease on grapevines.
The objective of this study was to design and validate an RT
quantitative real-time PCR (RT-qPCR) assay, to efficiently quantify
GFLV virus in grapevine tissues through the whole season. For
method validation total RNA was isolated from grapevine material
containing the reference GFLV isolate F13. LOD and LOQ for the
designed one-step RT-qPCR assay were evaluated by testing 10fold serially diluted RNA (from undiluted to 10-8) in triplicate (intraassay) in three independent runs (inter-assay). The dilutions
whose Cq values from three separate runs gave coefficient of
variation (CV) ≤ 30% were assumed to be within the range of
quantification (Burns et al., 2004). Theoretical genome copy
concentrations per reaction were assigned, taking into account
that the manifestation of stochastic effects (not all reactions in
triplicate detected or a high intra- or inter-assay CV) is indicative of
the presence of ≤ 10 target copies per reaction (Ellison et al.,
2006). The results showed that reliable quantification could be
performed within the range from 102 to at least 107 genome copies
per reaction. Phloem, which is the toughest grapevine material for
downstream processing and analysis, was sampled from six
grapevines in Slovenia at four seasonal time (June, July, August
and September). RNA was isolated and 10-fold and 100-fold
dilutions tested in duplicates with the RT-qPCR assay for GFLV
plus two reference gene assays (COX and 18S). Both reference
genes were compared using geNorm software (Pfaffl, 2001;
Vandesompele et al., 2002), obtaining an M value of 0.488. This
indicates a high seasonal correlation and stability of the COX and
18S expression, which, together with the fact that both genes
belong to different metabolic pathways, makes them suitable
normalisers for GFLV relative quantification.
Relative
quantification was expressed as a ratio (r) of the GFLV
concentration in each sample to the GFLV concentration in a
defined calibrator sample and normalised to the reference gene
expression, which was represented by the geometric mean of both
reference genes (Vandesompele et al., 2002). The relative
expression ratio was calculated based on the amplification
efficiencies of both GFLV and the reference genes, E=10(1/slope)
(where the slope means DCq between 10- and 100-fold dilutions)
(Pfaffl, 2001). The inclusion of amplification efficiencies together
with the reference gene normalisation minimized potential
intersample variations due to differences in the efficiency of RNA
isolation, reverse transcription and the amplification itself. The r
values were proportional to the GFLV genome concentration and
showed seasonal fluctuations of GFLV concentration in phloem
with the lowest amount of viral RNA being found during the
summer in the majority of the tested grapevines.
P022
Butyrivibrio fibrisolvens strains are presently recognized as the
major butyrate-producing bacteria found in the rumen. They can
be found in the digestive track of many animals and also in the
human gut. Aim of this study was to develop a powerful
quantitative competitive polymerase chain reaction (QC-PCR)
assay based on 16S rDNA for the enumeration of the strains
belonging to Butyrivibrio fibrisolvens. Species-specific PCR
primers used to amplify partial 16S rDNA region (213 bp as target
DNA) were chosen from the literature. Two internal primers,
bearing 5' tails which contain two ~30 nucleotide sequences which
are unrelated to the target to be amplified and complementary to
each other, were designed. To construct the homologous
competitor with 50 bp insertion a stepwise SOE-PCR (Splicing by
Overlap Extension Polymerase Chain Reaction) in three separate
amplifications using different primer pairs was carried out. For
several future applications and as the easiest way to determine
competitor concentration, competitor fragment was cloned into a
TA plasmid vector (pTZ57R/T) and was purified and quantified
before used. Competitor was serially diluted and co-amplified by
PCR with total extracted DNA from rumen fluid samples. QC-PCR
products were electrophoresis on agarose gel containing ethidium
bromide, and photographed. Band intensities were measured
using image analysis software (imageJ 1.42q) to determine if coamplification occurred with equal efficiency. Plotting the quantity of
competitor against the ratio of amplified target to amplified
competitor using log scale was evaluated by simple regression by
using JMP software and finally R2 was estimated as a criterion of
competitive PCR performance. The log plot of the amount of
amplified target DNA against the amount of amplified competitor
DNA was highly linear (R2 = 0.985). As a result of our study a new
competitive PCR assay was developed for quantification and
enumeration of Butyrivibrio fibrisolvens in rumen fluid samples.
Submicroscopic duplications identified during
prenatal aneuploidy testing
P024
Alison Hills1, Kathy Mann1, Caroline Mackie Ogilvie2
1
GSTS Pathology, Guys and St Thomas NHS Foundation Trust,
United Kingdom; 2Cytogenetics Department, Guys and St Thomas
NHS Foundation Trust, United Kingdom
A Modular approach in method validation of the realtime PCR module for the detection of five VTEC
Escherichia coli serogroups
Rapid semi-quantitative QF-PCR (Quantitative Fluorescence
PCR) is a widely-used approach for the prenatal diagnosis of
viable autosomal aneuploidies. Since 2000, we have tested
>40,000 prenatal samples using a one-tube multiplex containing
seventeen polymorphic microsatellite markers specific for
chromosomes 13, 18 and 21 to diagnose trisomy for these
chromosomes. During this period, 67 (~0.15%) samples exhibited
a triallelic profile at a single locus, with all other flanking loci
showing normal biallelic or uninformative results. Such findings
may indicate either benign submicroscopic duplications (SMDs),
or partial chromosome imbalance with phenotypic consequences.
Five recurring SMD regions were identified on chromosome 13,
six on chromosome 18 and three on chromosome 21. In all but
one case, QF-PCR analysis of parental blood samples showed
inheritance of the SMD, indicating benign variation; however, the
additional testing required to establish this is likely to cause
parental anxiety. In view of this, CMGS/ACC Best Practice
Guidelines now recommend that SMDs that have previously been
associated with a normal parental phenotype are not reported;
markers that identified the most frequent of these have been
replaced in our multiplex. However, previously unreported SMDs
that are not present in the database of genomic variants present
difficulties of interpretation, especially when found to be de novo,
or to represent terminal or unflanked loci. It is therefore important
that the incidence and aetiology of these SMDs is made available.
The inherited and de novo SMDs identified in our laboratory will be
presented. One SMD region on chromosome 13 was investigated
using quantitative FISH and PCR; the duplicated segment was
found to be between 20 and 130 kb in length and appeared to be
tandem and of a similar size in all tested cases.
P023
The Design and Construction of Competitor Fragment
by SOE-PCR for Quantification of The Ruminal
Bacterium: Butyrivibrio fibrisolvens
Amir Taheri Ghahfarokhi, Mojtaba Tahmoorespur, Mohammad
Reza Nassiry, Mohammad Hadi Sekhavati
Ferdowsi University of Mashhad, Mashhad, Iran
Dafni-Maria Kagkli1, Thomas P. Weber1, Marc Van den
Bulcke1, Silvia Folloni1, Stefano Morabito2, Rosangela
Tozzoli2, Monica Ermolli1, Laura Gribaldo1, Guy Van den Eede1
1
1European Commission Joint Research Centre, Institute for
Health and Consumer Protection, Molecular Biology and
Genomics Unit, via E. Fermi 2749, I-21027, Ispra (VA), Italy;
2
Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161,
Rome, Italy
The detection and identification of the presence of pathogenic
micro-organisms in products often requires the simultaneous
demonstration of distinct DNA sequences in a single viable
organism. While the latter can only be demonstrated with classical
microbial assays, combined PCR analysis allow for the very
sensitive detection of multiple pathogenic sequences in a sample
(be it DNA or RNA). To support concise decisions, the applied
PCR methods should however exhibit similar performance for a
number of criteria (Bustin et al., 2009). In various fields where
PCR is applied, the method specificity and sensitivity are generaly
recognized as the minimal critical performance parameters. Here,
we demonstrate the results of the assessment of a set of real-time
PCR methods proposed to CEN (CEN TC275/WG6) for the
detection of Verocytotoxin Escherichia coli (VTEC). While the ISO
standard 16654:2001 is focused only on the detection of E. coli
O157 serogroup, we have extended the scope to a number of
other important serogroups (O26, O103, O111, and O145).
Specificity of the methods was demonstrated in silica by
bioinformatics analysis and experimentally. The dynamic range
and the PCR efficiency of all methods was determined by
standard curve dilution. Finally, the limit of detection of each
method was determined by serial dilution analysis. All PCR
methods apply TaqMan probe-based detection, targeting the two
E. coli toxin genes, their variants (vtx1, vtx2), and the intimin (eae)
gene. All methods were optimized and assessed against the
criteria set by the EURL-GMFF for real-time PCR methods
(http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm). All methods
perform adequately: all are highly specific to the expected targets,
exhibit a linear correlation between 10 to 10.000 copies, have an
LOD ranging from 10 to 20 copies and a PCR efficiency of >90 %
(except for the methods for O103 and O145). Our data
demonstrate that this set of VTEC detection/identification methods
represent a valuable series of PCR methods that can be combined
into a reliable VTEC decision support system. The applied socalled 'modular approach' in the method performance assessment
will be discussed.
Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, Jim
Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W. Pfaffl,
Gregory L. Shipley, Jo Vandesompele, and Carl T. Wittwer (2009) The
MIQE Guidelines - Minimum Information for Publication of Quantitative
Real-Time PCR Experiments. Clinical Chemistry. 55(4): 611-622
P025
based on SYBR Green chemistry were found to be optimal for the
assay. Kinetic studies pinpoint an upregulation in carB and carRA
genes involved in carotene biosynthesis which extends from an
early growth phase of 24hours upto 144hours (6days).Contrarily,
the expression pattern of tsp3 and tsp1 involved in β–carotene
catabolism remain stable throughout. For ensuring reliable
conclusions, we are investigating 3 more reference genes namely,
gpd (Glyceraldehyde phosphate dehydrogenase), EF-1α
(translation elongation factor 1-alpha) and pyrG (orotidine- 5’monophosphate decarboxylase) for data analysis and
normalization using the software Qbase.
Comparative study of direct sequencing and
TheraScreen assay for KRAS mutational analysis
P027
LUZ MARTINEZ AVILES, MAR IGLESIAS, ESTER MORAGON,
ERICA TORRES, GEMMA NAVARRO, MAR GARCIA GARCIA,
SERGI SERRANO, BEATRIZ BELLOSILLO
HOSPITAL DEL MAR, Spain
Development and evaluation of a one-step real-time
RT-PCR assay for universal detection of influenza A
viruses from avian and mammal species
BACKGROUND: Epidermal growth factor receptor inhibitor
therapy is used for treatment of metastatic colorectal carcinomas
(CRC) and non small cell lung cancer (NSCLC) in patients lacking
KRAS mutations. Patients carrying mutations in the KRAS gene
do not respond to this therapy. AIM: The aim of this study was to
compare the analytical sensitivity of two different procedures to
detect KRAS mutations. METHODS: We have determined the
mutational status of the KRAS gene in tumoral samples from 151
patients with CRC and 13 from NSCLC patients by two
methodologies: 1. PCR amplification of exon 2 of the KRAS gene
followed by direct sequencing using BigDye 3.1 and analysis in a
genetic analyzer (Applied Biosystems) and 2. TheraScreen (DxS)
assays using a real time PCR system (Roche Diagnostics).
RESULTS: Analyzing KRAS in 151 biopsies of CRC patients we
observed a 98% of concordance between both techniques. Ninetynine patients were negative for KRAS mutations in codons 12 or
13. Forty-seven patients presented mutations in one of these two
codons, but 6 out of 47 cases showed doubtful sequencing
results, so the TheraScreen assay was determinant to establish
the final positivity of these cases. Moreover, the TheraScreen
assay detected three positive cases which direct sequencing had
failed to detect due to the low mutant allele burden. In addition, we
analysed 13 patients with NSCLC of which 10 showed ambiguous
sequencing results so the TheraScreen assay was needed to
confirm the mutations. Finally, four cases presented by direct
sequencing, infrequent KRAS mutations (G13R, G13C and
G12F). The TheraScreen assay was not able to detect the two
mutations affecting codon 13 because they are not included in the
test. Regarding the G12F mutation, although it is not included in
the assay, it showed an unspecific positivity for the G12V
mutation. CONCLUSIONS: Both direct sequencing and
TheraScreen assayshowed a good correlation although
TheraScreen assay has a greater sensitivity than direct
sequencing in samples with low mutant allele burden.
TheraScreen assay may be, in some cases, less specific than
direct sequencing and additional mutations should be included in
the test in order to avoid false negative results.
P026
Developing an optimal qRT-PCR strategy for fungal
transcriptomics on carotenoid metabolism
Yamuna Sahadevan1, Wilhelm Boland1, Kerstin Hoffmann2,
Kerstin Voigt2
1
Dept.of Bioorganic Chemistry, Max Planck Institute for Chemical
Ecology,Jena, Germany; 2Jena Microbial Resource Collection,
Dept.of Microbiology and Molecular Biology,University of Jena and
Hans-Knöll-Institute, Jena,Germany
In zygomycetous fungi, β–carotene production is augmented
several folds in mated cultures rather than in individual mating
partners. Despite the fact that a cocktail of trisporic acid sex
hormones induce carotenogenesis, little is known about the link
between these two factors at the genetic level. We aim to develop
a reliable qRT-PCR method following MIQE guidelines for the
mRNA quantification of fungal genes involved in carotenoid
metabolic pathway. Mating experiments with Blakeslea trispora
were done for kinetic analysis of 4 target genes using act-1(actin)
as the reference gene for relative quantification. A standard curve
was used to determine the reaction efficiency of primers based on
a 5-fold dilution series, using in-built Stratagene Mx3000P
software. A high annealing temperature of 60° C with 35 cycles
Alexander Nagy1, Veronika Vostinakova1, Zuzana
Pirchanova1, Lenka Cernikova1, Zuzana Dirbakova2, Miroslav
Mojzis2, Helena Jirincova3, Martina Havlickova3, Adam Dan4,
Krisztina Ursu4, Stefan Vilcek5, Vlastimil Krivda1, Jitka
Hornickova1
1
State Veterinary Institute Prague, National Reference Laboratory
for Avian Influenza and Newcastle Disease, Czech Republic;
2
State Veterinary Institute Zvolen, National Reference Laboratory
for Avian Influenza and Newcastle Disease, Zvolen, Slovak
Republic; 3National Institute of Public Health, National Reference
Laboratory for Human Influenza, Prague, Czech Republic;
4
Central Agricultural Office, Veterinary Diagnostic Directorate,
Molecular Biology Laboratory, Budapest, Hungary; 5University of
Veterinary Medicine in Kosice, Department of Parasitology and
Infectious Diseases, Kosice, Slovak Republic
The objective of our study was to develop and evaluate a TaqMan
real-time RT-PCR (RRT-PCR) assay for universal detection of
influenza A (IA) viruses. The primers and LNA-modified
octanucleotide probe were selected to correspond to extremely
conserved regions of the membrane protein (MP) segment
identified by a comprehensive bioinformatics analysis including
10,405 IA viruses MP sequences, i.e., all of the sequences of the
Influenza Virus Sequence database collected as of August 20,
2009. The RRT-PCR has a detection limit of approximately five
copies of target RNA/reaction and excellent reaction parameters
tested in four IA viruses reference laboratories. The inclusivity of
the assay was estimated at both the bioinformatic and the
experimental level. Our results predicted that this RRT-PCR assay
was able to detect 99.5% of known human IA virus strains,
99.84% of pandemic influenza A (H1N1) strains, 99.75% of avian
strains, 98.89% of swine strains, 98.15% of equine strains, and
100% of influenza A viruses of other origin.
P028
Dietary fibers are not born equal: qPCR to define
prebiotic properties
F DEPEINT, G ABDEL NOUR, CN NIAMBA, P POUILLART, A
ABDEL NOUR
Prebiotics have been defined by Roberfroid et al (2004) as
selectively fermented ingredients that allow specific changes, both
in the composition and activity of the gastrointestinal microflora
that confer benefits upon host well being and health. Most
researchers consider that changes in the composition of gut
microflora can be characterised by an increase in probiotic
bacteria (eg. Bifidobacterium, Lactobacillus) and/or decrease in
pathogenic bacteria (eg. Bacteroides, Clostridium). Similarly,
changes in the bacteria activity is often characherised by a
butyrogenic activity. We present here two clinical trials
investigating prebiotic potential of dietary oligosaccharides. In
each of those studies Bifidobacterium and Clostridium were
amplified using qPCR and quantified against standard curves.
P029
Disrupted balance between phase I and phase II
estrogen-metabolising enzymes may contribute to the
growth of endometriotic tissue in ovarian
endometriosis
Neli Hevir1, Martina Ribič-Pucelj2, Tea Lanišnik Rižner1
1
Institute of Biochemistry, Faculty of Medicine, University of
Ljubljana, Ljubljana, Slovenia; 2Department of Obstetrics and
Gynaecology, University Medical Centre, Ljubljana, Slovenia
Endometriosis is a complex estrogen-dependent disease defined
as the presence of endometrial tissue outside the uterine cavity.
Estrogens stimulate cell proliferation via the estrogen receptor and
undergo extensive oxidative metabolism at different positions,
catalyzed by various cytochrome P450 isoforms. Formation 2- and
4-hydroxy-estrogens (catechol estrogens, CEs), is catalysed
mainly by CYP1A1/1A2 and CYP1B1, respectively. CEs can be
oxidized to the corresponding estrogen ortho-quinones with
concomitant formation of the reactive oxygen species (ROS).
NAD(P)H:quinone oxidoreductase (NQO1/2) regulate the
reduction of toxic estrogen quinones back to catechols.
Furthermore, estrogens can undergo 16α-hydroxylation,
extrahepaticaly catalysed mainly by CYP3A5 and CYP3A7. CE
and 16α-hydroxy-estrogens are further metabolised by the
conjugative enzymes: catechol-O-methyltransferase (COMT),
sulfotransferases
(SULTs),
UDP
glucuronosyltransferases
(UGTs), and glutathione S-transferases (GSTs) and yields less
harmful products. Expression levels of genes encoding phase I
(CYP1A1, CYP1A2, CYP1B1, CYP3A5, CYP3A7) and phase II
(SULT1A1, SULT1E1, SULT2B1, COMT, UGT2B7, NQO1, NQO2,
GSTP1) estrogen-metabolizing enzymes were studied by real-time
PCR in 31 samples of ovarian endometriosis and 29 normal
endometrium samples. PPIA, GAPDH, and RPLP0 were used for
normalisation. MIQE guidelines were considered in the
performance and interpretation of the qPCRs. We found
significantly higher levels of CYP1A1 and CYP3A7 (p < 0.0001)
suggesting increased 2- and 16α-hydroxylation of estrogens.
CYP1B1 and CYP3A5 expression was not altered, while CYP1A2
expression was not detected. Among conjugative enzymes, we
found increased mRNA levels of COMT (p < 0.0001) and
unchanged mRNA levels of SULT1A1 and NQO2, while all the
others gene levels were significantlly decreased: UGT2B7, NQO1,
GSTP1, SULT2B1 (p < 0.0001), and SULT1E1 (p = 0.0311). Our
qPCR data thus suggest increased phase I and decreased phase
II metabolism of estrogens that leads to exessive ROS formation,
which may activate cytokines that control the implantation and the
growth of endometrial cells outside the uterus and thus contribute
to the development of ovarian endometriosis.
P030
Evaluation of Various Concentration Methods for
Detecting Enterovirus in Water
Bing-Mu Hsu
National Chung Cheng University, Taiwan, Republic of China
Enteroviruses generally spread through the fecal-oral route, but
the importance of viral transmission by water is probably
underestimated. A reliable, sensitive, and practical method for
detecting small concentrations of enteroviruses is needed. The
objective of this paper is to evaluate series of procedures to
concentrate enteroviruses by adsorption to and elution from
various charged membranes for various types of water. The
relative recovery efficiencies of enterovirus in treated water
samples were also evaluated by Real-Time PCR. The optimum
procedures designed for enteroviruses detection of the
environmental water samples were done in this study. Two stage
processes, prefiltration and adsorption, were used for separating
enteroviruses from large volume of water sample. The water
samples passed through prefilter apparatus were collected, and
then were filtered through the evaluated membranes for
adsorption. The types of adsorbing disc filters evaluated in this
study includes positive charged membrane (6408502 1MDS, 47mm diameter, CUNO Inc., Meriden, Conn. U.S.A.), negative
charged membrane (NM04701 020SP, 47-mm diameter, CUNO
Inc., Meriden, Conn. U.S.A.), and nitrocellulose membrane (HA
series, 47-mm diameter, 0.45-um pore size, Millipore, Ireland).
The relative recovery efficiencies of various concentration
methods for enteroviruses were analyzed by Real-Time PCR and
shown in the Table 1. The optimum procedure is followed as that
the water sample was prefiltered first. The prefiltered water was
adjusted pHs and electrolyte, then passed through nictrocellulose
membrane. Aliquot of H2SO4 was passed through the membrane
to rinse out the cation, and then NaOH was poured on the
membrane and the filtrate was recovered in a tube containing
H2SO4 and TE buffer for neutralization. All of the eluate was
concentrated with an ultrafiltration system. The contents of
environmental inhibitors after virus concentration are suspected to
have some inhibitory effect on viruses PCR detection. It should be
considered while detecting enteroviruses in environmental
samples.
P031
Event-specific detection of genetically modified rice
(Kefeng 6) by means of quantitative real-time PCR
Patrick Guertler, Ingrid Huber, Sven Pecoraro, Ulrich Busch
Bavarian Health and Food Safety Authority, Germany
Rice is one of the most important food crops in the world,
especially in Asian countries. However, heavy losses in rice yield
(24%-41% per year) are caused by rice insect pests. To address
this issue, several varieties of genetically modified (gm) rice have
been developed and released for field trials in China. One crop
under study is the insect resistant rice called “Kefeng 6”. This
transgenic rice line has been altered by insertion of genes
encoding for Cry1Ac and CpTi, both proteins responsible for an
increase insect resistance. The hygromycin resistance gene (hpt)
has been inserted as a selective marker. Despite the fact that gm
rice has not been approved for commercial cultivation in China (or
elsewhere in the world), there were reports on illegal planting and
trade of gm rice in a central province of China. By way of the
global trade, Kefeng 6 rice was found in food samples in the
Netherlands in 2010 (rapid alert system for food and feed, RASFF
No. 2010.0336). In the European Union (EU), Kefeng 6 is not
authorized for import, processing or cultivation and material
containing Kefeng 6 is therefore not permitted at any level. In
order to monitor the potential presence of Kefeng 6 rice in
products imported into the EU, a sensitive and specific eventspecific detection method needs to be on hand to clearly
distinguish between different gm rice lines. Therefore, it was the
aim of this study to develop an event-specific detection method for
the gm rice Kefeng 6 by using quantitative real-time PCR (qPCR)
and to validate this method according to the guidelines of GMO
testing. Afterwards, this method was used to monitor several rice
products on a small scale level. Based on the flanking sequence
of the DNA insert, primers and a hydrolysis probe were designed
and used in a qPCR assay applying the ABI TaqMan technology.
The assay was validated as outlined in the “Minimum performance
requirements for analytical methods for GMO testing” and is
presented as outlined in the MIQE guidelines. The validation
process revealed a limit of detection of 5 copies of the transgene
with confidence intervals (95%) between 0.07 and 0.52, a mean
PCR efficiency of 105 % and a mean R2 of 0.99. The specificity
was determined by analysis of different gm and non-gm crops by
using this assay. No positive amplification signal was observed for
any of the analyzed crops. Furthermore, the assay was tested by
analysis of rice samples obtained on the local market. Only one
sample, that has been previously tested positive by means of a
construct-specific detection assay, was again tested positive by
using this assay. This underlines the suitability of this assay for the
monitoring of food samples for the presence of Kefeng 6 rice.
P032
Examining resistance gene expression using reverse
transcriptase qPCR
Tine Yding Wolff1, Jan Lorenzen1, Trine Rolighed Thomsen1,2,
Anne Karin Ildor Rasmussen3, Henrik Stender3, Helle
Stendahl Andersen1, Masumeh Chavoshi1
1
The Danish Technological Institute, Denmark; 2Aalborg
University, Denmark; 3AdvanDx, Denmark
Objective: The aim of the study was to examine the expression of
the mecA gene in clinical methicillin-resistant Staphylococcus
aureus (MRSA) isolates upon induction with cefoxitin using
reverse transcriptase qPCR (RT-qPCR). Methods: 18 S. aureus
strains (16 MRSA representing staphylococcal cassette
chromosome mec (SCCmec) types I-V and 2 MSSA) were grown
with shake at 35°C for 40 min with and without cefoxitin.
RNAprotect Bacteria Reagent (Qiagen) was added to 1 mL
suspensions of each culture and the cells were disrupted by
enzymatic (lysostaphin) and mechanical (bead beating) lysis. Total
RNA was extracted using the RNeasy PLUS mini kit (Qiagen)
including the optional DNAse treatment step. cDNA was
synthesized from 100 ng RNA using the SuperScriptTM III First
Strand Synthesis System with random hexamers (Invitrogen). The
cDNA was template in qPCR reactions targeting the mecA and the
16S rRNA genes respectively. On basis of the qPCR data the
baselines for cefoxitin induced and uninduced cells and the mean
fold changes (MFC) in mecA expression were calculated as
described by Shang et al. (2010). The procedure was carried out
three times for each strain and to evaluate the effect of induction
on mecA expression uninduced and induced baselines were
compared by paired Students’ t-tests. The minimum inhibitory
concentration (MIC) of cefoxitin was determined for each isolate
using E-test. Results: Significant changes in the expression of
mecA upon cefoxitin induction (P<0.05) were observed for seven
of the MRSA isolates representing three different SCCmec types
(type II (n=2), type IV (n=3) and type V (n=2)). Throughout the
study no correlation between the SCCmec type and the level of
mecA expression was observed. The cefoxitin MICs of the MRSA
isolates ranged from 12 to 256 µg/mL and there was a tendency
for strains with a low cefoxitin MIC to have a low mecA expression
as well. Conclusion: It was possible to apply RT-qPCR for the
detection of changes in the mecA expression upon cefoxitin
induction. Significant changes (P<0.05) were observed for seven
of the examined MRSA isolates. There was no correlation
between the SCCmec type and the induced baselines, but it
seemed that isolates with a low cefoxitin MIC also had a low
expression level of mecA.
Shang et al, Antimicrob Agents Chemother 2010, p. 956-59.
P033
Experience with Plant RNA Isolation Aid for the total
RNA isolation from plants
Irena Mavric Plesko1, Mojca Virscek Marn1, Natasa Toplak2,
Minka Kovac2
1
Kmetijski institut Slovenije, Slovenia; 2Omega d.o.o., Slovenia
Plant tissue is known as a difficult tissue for RNA extraction since
it can contain big amounts of secondary metabolites, such as
polysaccharides and phenolics. Some of these compounds are
known PCR inhibitors. We investigated the influence of Plant RNA
Isolation Aid (Life Technologies) on quality and quantity of
extracted total RNA (totRNA) from different plant tissue. TotRNA
was extracted on MagMAX™ Express Magnetic Particle
Processor using MagMAX™-96 Total RNA Isolation Kit (Life
Technologies) with and without addition of Plant RNA Isolation
Aid. The analyzed plant tissues included raspberry leaves, in vitro
potato plantlets, potato tubers, grapevine leaves and tomato
leaves. Extracted totRNA was reverse transcribed and its quantity
was checked using 18S rRNA qPCR assay. The results of qPCR
show no differences when Plant RNA Isolation Aid was used for
totRNA extraction from potato and tomato. With raspberry and
grapevine tissue the differences in Ct values were between 3 and
19. The results of our study show that Plant RNA isolation Aid can
improve RNA extraction from certain plant species.
P034
Expression of Laccase genes in Schizophyllum
commune
Soumya Madhavan, Katrin Krause, Erika Kothe
Friedrich Schiller University, Institute of Microbiology, Microbial
Phytopathology, D-07743 Jena, Germany
Schizophyllum commune, a saprophytic white rot fungus, is
involved in the degradation of complex organic molecules
including lignin. Previous reports say that this fungus can degrade
refractory organic matter from black slate with the help of different
exoenzymes which perform radical reactions to oxidize organic
matter. The genome of S. commune reveals 16 FOLymes (Fungal
Oxidative Lignin Enzymes), which are potentially involved in lignin
degragation. These genes include three lignin oxidases (LO) and
13 lignin degrading auxiliary enzymes (LDA) genes. In this study,
relative expression of the laccase and laccase-like genes (LO
family) in S. commune areinvestigated using quantitative real time
PCR (RT-PCR). Two laccases and four laccase-like genes were
selected for expression studies based on microarray data
comparing S. commune grown on a black slate surface and grown
on complex yeast medium. Also, the involvement of laccases in
hyphal morphogenesis, especially during fruitbody formation is
investigated by analyzing the relative expression of the genes of
interest in hyphae and fruitbody tissue. Preliminary results reveal
that laccase expression is upregulated in the cultures with black
slate powder. This shows that laccase and laccase-like genes are
influenced by the presence of black slate in minimal medium
which is to be expected if laccases are involved in the degradation
of stone material.
P035
Expression of local luteotropic factors during induced
luteolysis in the bovine corpus luteum
Dieter Schams1, Heinrich Meyer1, Bajram Berisha1,2
1
Germany; 2Faculty of Agriculture and Veterinary, University of
Prishtina, Kosovo
The essential role of endometrial prostaglandin F2 alpha (PTGF)
for induction of the corpus luteum (CL) regression is well
documented in the cow. However, the acute effects of PTGF on
known local luteotropic factors: oxytocin (OXT) and its receptor
(OXTR), insulin-like growth factor 1 (IGF1), and progesterone
(PG) and its receptor (PGR), were not thoroughly studied in detail.
The aim of this study was therefore to evaluate the tissue
concentration of these factors during PTGF induced luteolysis. In
addition the mRNA expression of PGR, OXTR, IGF1, insulin-like
growth factor binding protein 1 (IGFBP1), was determined at well
defined times after PTGF treatment. Cows (n=5 per group) in the
mid-luteal phase (Days 8–12, control group) were injected with the
PTGF analog (cloprostenol), and CL were collected by
transvaginal ovariectomy at 0.5, 2, 4, 12, 24, 48, and 64 h after
injection. The mRNA expression was analyzed by quantitative
real-time RT-PCR, and the protein concentration in tissue was
evaluated by enzyme immunoassay or radioimmunoassay. PG
concentrations, as well as mRNA expression of PGR, in CL tissue
were significantly down regulated by 12 h after PTGF. Tissue OXT
peptide and OXTR mRNA decreased significantly after 2 h,
followed by a continuous decrease of OXT mRNA. IGF1 protein
already decreased after 0.5 h. By contrast, the IGFBP1 mRNA
was up-regulated significantly after 2 h to a high plateau. The
acute decrease of local luteotropic activity may be a key
component in the cascade of events leading to functional
luteolysis in the cow.
P036
Expresssion of Adrenomedullin signalling factors in
bovine corpus luteum during oestrous cycle,
pregnancy and induced luteolysis
Bajram Berisha1,2, Stefanie Schilffarth1, Mihir Sarkar1, Dieter
Schams1
1
Germany; 2Faculty of Agriculture and Veterinary, University of
Prishtina, Kosovo
Investigations on "nonclassic" regulators of angiogenesis could
open new perspectives in understanding angiogenesis under
physiological and pathological conditions. A pro-angiogenic effect
has been demonstrated for adrenomedullin (AM), it´s co-factor
activity-modifying protein 2 (RAMP-2) and the Calcitonin receptorlike Receptor (CALCLR). This signalling pathway is besides
associated with lymphangiogenesis, that (in a physiologically way)
is still only poorly understood in ovary and its structures. The aim
of this study was therefore to evaluate mRNA expression of AM,
RAMP-2 and CALCLR in bovine corpora lutea (CL) during
different physiological stages. Experiment 1: CL were assigned
days 1-2, 3-4, 5-7, 8-12, 13-16, >18 (after regression) of oestrous
cycle and of gravidity (month <3, 3-5, 6-7 and >8). Experiment 2:
Induced luteolysis. Cows on days 8-12 were injected with a
PGF2α analogue and CL were collected by transvaginal
ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2α
injection. Tissue levels of mRNA were characterized by real-time
RT-PCR. All 3 factors were clearly expressed. AM and CALCRL
showed significant changes in both experiments, RAMP-2 during
induced luteolysis. For AM highest levels could be observed at the
beginning and the end of the luteal phase, CALCRL remained at
comparable levels. Both factors showed their highest expression
at the end of pregnancy. After induced luteolysis the expression of
all factors started to decline (significantly during structural
luteolysis). In conclusion, our results could lead to the assumption
that factors investigated may be involved in mechanisms
regulating CL formation, function and regression – with a special
emphasis during the final months of pregnancy.
P037
Gene Expression of Selected WRKY Transcription
Factors in Arabidopsis thaliana Cell Cultures during a
Parabolic Flight
Anne Hennig, Maren Neef, Niklas Hausmann, Rüdiger Hampp
University of Tuebingen, Physiological Ecology of Plants,
Germany
Plants use gravity as a guide for growth and development. Recent
studies could demonstrate that plant cells which are not part of
specialized tissues such as the root columella cells are also able
to sense gravitational forces. Therefore we used undifferentiated,
homogeneous cell cultures of Arabidopsis thaliana (cv. Columbia)
(A.t.) in order to identify early alterations in gene expression as a
response to altered gravitational fields. As experimental system
we used parabolic flights (A300, Novespace, France). A single
parabola consists of following phases: 1g, 1.8g for 20 sec,
microgravity (µg) for 22 sec, 1.8g for 20 sec, 1g. Up to 30
parabolas are exercised within one flight. By the use of
microarrays (ATH1, Affymetrix) we found several hundred
transcripts to be altered in amount during the respective gravity
segments. For this study, we specifically looked for genes at the
beginning of signal transduction chains, such as transcription
factors (TFs). TFs are small proteins regulating gene expression
of their target genes by binding to specific promoter sequences.
For quantification via qRT-PCR, six WRKY genes were selected.
WRKYs are known to be involved in various physiological
processes such as senescence or pathogen defense. The data
indicated a transient gene expression profile, while most of the
analyzed WRKYs were significantly down-regulated after the
microgravity-phase of the parabola.
P038
Gene expression studies on soil fertility and nutritional
status in grapevine
Nadia Bertazzon1, Piergiorgio Stevanato2, Alessandro Zonin2,
Giuseppe Concheri2, Massimo Saccomani2, Elisa Angelini1
1
C.R.A. Centro di Ricerca per la Viticoltura, 31015, CONEGLIANO
(TV), Italy; 2Università degli Studi di Padova, Dipartimento di
Biotecnologie Agrarie, 35020, LEGNARO (PD), Italy
Grapevine is a major fruit crop worldwide that is susceptible to
several environmental stresses like diseases and water-nutritional
deficiency. Its management involves many agronomic practices
and phytochemicals to prevent significant reductions in quality and
quantity of production caused by these stresses. There is an
urgent need for developing alternative sustainable strategies for
grapevine protection. This requires innovative and interdisciplinary
approaches for a better understanding of grapevine responses to
environmental stresses. The aim of this study was to evaluate the
relationships among grapevine productivity, main soil chemical
properties and expression of genes that could be involved in
defence mechanism of grapevine to edaphic stresses. The
research was conducted in two vineyards characterized by high
and low productivity, both cultivated with cv. Garganega on 1103
Paulsen and located in the North-East of Italy. The evaluation of
the soil properties revealed large and significant differences
(p<0.001) between the two vineyards. The soil of vineyard
characterized by high productivity showed neutral pH, good supply
of organic matter and appropriate C/N ratio (8:1). On the contrary,
the soil of less productive vineyard was characterized by acid pH,
scarce organic matter content, suboptimal C/N ratio and leave
content of nitrogen and sulphur. Four selected defence-related
genes were analysed by qPCR starting from total RNA of
grapevine leaves collected on August 2010 from the two
vineyards. Two genes, phenylalanine ammonia lyase (PAL) and
stilbene synthase 1 (VST1), encode key enzymes of the
phenylpropanoid pathway which leads to the production of various
defence-related compounds, like resveratrol. Another gene
encodes the enzyme sucrose synthase (Susy) which is involved in
the metabolism of sucrose. The last gene encodes for a WRKY
transcription factor, implicated in the regulation of transcriptional
reprogramming associated with plant immune responses. A set of
five commonly used Vitis vinifera reference genes, encoding actin
(ACT), cytochrome oxidase (COX), pyruvate dacarboxilase (PDC),
glyceraldehyde-3-phosphate dehydrogenase (GPDH) and 26S
ribosomal RNA (26S), were used to normalize expression data.
Two genes, GAPDH and ACT, were selected among them for the
normalization process, as they showed stable expression in all our
experimental conditions. All the target genes analyzed showed
significant higher relative expression values (p<0.001) in plants
from low-productive vineyard with respect to those from highproductive vineyard. These results showed that deficiencies in soil
fertility and nutritional state of grapevine lead to the induction of
the selected defence-related genes which could be involved in
phenotypic adaptation of vine plants to edaphic stress. Further
qPCR experiments will be carried out for the validation of these
gene expression patterns on nitrogen and sulphur-starved plants
grown under controlled conditions.
P039
H275Y screening and quantification using isolated
unlabeled probe
Hong Kai Lee1, Chun Kiat Lee1, Julian Wei-Tze Tang1, Evelyn
Siew-Chuan Koay1,2
1
Molecular Diagnosis Centre, Department of Laboratory Medicine,
National University Hospital, Singapore; 2Department of
Pathology, National University of Singapore, Singapore
Objectives: An increasing number of oseltamivir-resistance
associated mutations (H275Y in the neuraminidase NA gene) are
being reported in H1N1/2009 viruses isolated from patients treated
with oseltamivir, particularly in the immunocompromised. 1 It is
important to identify the presence of these drug resistanceassociated mutations to optimize the care for such patients.
Methodology: A rapid and cost-effective High Resolution Melting
(HRM) PCR assay was developed for this purpose using an
unlabeled probe to screen for the H275Y mutation among patients
treated with oseltamivir, especially the highly susceptible
immunocompromised patients. Isolated probe PCR (IP-PCR)2 was
performed using Superscript III Platinum One-step qRT-PCR kit
(Invitrogen) on either the LightScanner LS32 (Idaho) or the
LightCycler LC v2.0 (Roche) system, both of which utilize the
uniquely designed LC glass capillaries as reaction receptacles. To
initiate the IP-PCR, 2 μL of extracted viral RNA was added to 6 μL
of reaction mix containing optimized amounts of forward and
reverse primers. A calculated excess amount of forward primer
and unlabelled probe mix (2 μL) was then aliquotted into the upper
well of the capillary tube, without being spun down with the
reaction mix. Upon completion of the IP-PCR, the 8 μL reaction
mix was mixed with the pre-deposited primer/probe mix by
inversion and reverse-spinning, followed by an ”extended” 15cycle asymmetric amplification reaction and continuous HRM data
acquisition. The in vitro synthesized RNA transcripts for both
wildtype and mutant were diluted using pooled negative RNA
extracts and used as the standard calibrator. The lower limit of
detection (LLOD) and lower limit of quantification (LLOQ) for the
mutant strain were determined. To estimate the proportion of
H275Y mutant population in a mutant/wildtype mixture, a series of
0-100% mixed-mutant/wildtype standards were tested, at 10%
incremental intervals and 105 RNA copies/reaction. Results: The
melt peaks for the unlabeled probe with a mismatched base C
(wildtype) and that with a perfectly matched base T (mutant) were
clearly discernable at 65.5°C and 69.0°C, respectively. The assay
reproducibly amplified concentrations of 20 to 2x107
RNAcopies/reaction, thus giving an LLOD of 20 copies/reaction.
The LLOQ, defined as the lowest concentration whereby the
difference of 3.32 Ct value per log is maintained, was 200
copies/reaction. Good resolution of the melt peaks was obtained
for the whole series of 0-100% mixed-mutant/wildtype standards.
Conclusion: This assay is able to provide easily discernable
results for qualitative and quantitative detection of WT
(H275)/mutant (Y275) mixes. The preliminary results suggest that
this assay and platform may accurately identify H275Y populations
down to 10% of the viral population, though further validation on
clinical specimens is required.
1. WHO, Wkly Epidemiol Rec 2010;85:37-40
2. MD Poulsen, CT Wittwer. Biotechniques 2007;43:87-91
P040
Improvement of phytoplasma diagnostics in grapevine
and fruit trees: from grinding to real time PCR analysis
Matevž Rupar, Nina Prezelj, Nataša Mehle, Petra Nikolić, Maja
Ravnikar, Marina Dermastia
National Institute of Biology, Slovenia
Phytoplasmas are plant pathogenic bacteria without cell wall and
may cause severe damage to their plant hosts. Examples of such
pathogens are Flavescence doreé (FD) and Bois noir
(BN)phytoplasma causing grapevine yellows and ‘Candidatus
Phytoplasma mali’ (AP), ‘Ca. P. prunorum’ (ESFY) and ‘Ca. P.
pyri’ (PD) from apple proliferation group phytoplasmas on fruit
trees. All of them can cause great crop losses and economical
damage if not detected soon enough and properly managed.
Because phytoplasmas cannot be grown in vitro, their diagnostics
depends only on molecular and serological methods. The most
commonly applied assays involve laborious and time consuming
steps of DNA extraction followed by the conventional PCR and
several PCR- RFLP analyses with agarose or polyacril amid gel
electrophoresis. Through recent years our laboratory developed
and improved methods for fast, Real time PCR based diagnostics
of phytoplasma in grapevine and fruit trees with higher sensitivity,
specificity and fewer possibilities for contaminations. We
compared the homogenization of plant tissues using mortar and
pistil in liquid nitrogen with a Fast Prep® homogenizer and
additionally compared both methods using real-time PCR
described below. For a DNA extraction CTAB method and
KingFisher® automatic extraction procedure based on magnetic
beads (Pirc et al., 2009) were compared with pipetting of a large
set of samples using an automatic pipetting workstation. The
PCR-PFLP analyses were compared with a real-time PCR assay,
using different primers and probes. We use primers and probes for
18S rRNA gene (Applied Biosystems, USA) as an internal control
of DNA isolation, primers and probes for universal phytoplasma
(Hren et al., 2007) detection phytoplasma and from apple
proliferation group (AP, PD, ESFY) (Nikolić et al., 2010). With a
new procedure, which combines a homogenization with a Fast
Prep® homogenizer, KingFisher® automatic extraction procedure
based on magnetic beads, pipetting of a large set of samples by
an automatic pipetting workstation and real-time PCR assays we
increased the number of processed samples in time, diminished
the possibilities of contaminations, improved the sensitivity and
specificity of detection and shortened the time for the diagnosis for
approximately three times. Therefore, new protocols combined
enables high throughput routine phytoplasma diagnostics.
Virus 1-Step Master Mix (Applied Biosystems by Life
Technologies) offers fast, sensitive and reliable (robust) detection
of norovirus genogroup II in comparision to the reagent from
another supplier.
1] Dingle, K.E., Lambden, P.R., Caul, E.O. and Clarke, I.N. 1995. Human
enteric Caliciviridae: the complete genome sequence and expression of
virus-like particles from a genetic group II small round structured virus. J
Gen Virol 76: 2349-2355.
2] Hoehne, M., Schreier, E. 2006. Detection of Norovirus genogroup I and II
by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe.
BMC Infectious Diseases 2006,
http://www.biomedcentral.com/1471-2334/6/69
3] Kageyama, T., Kojima, S., Shinohara, M., Uchida, K., Fukushi, S.,
Hoshino, F.B., Takeda, N., Katayama, K. 2003. Broadly reactive and highly
sensitive assay for Norwalk-like viruses based on real-time quantitative
reverse transcription-PCR. J Clin Microbiol, 41 (4): 1548-1557.
P042
Interest of Real-Time PCR for the Diagnosis of Invasive
Aspergillosis
Inès Hadrich1, Fattouma Makni1, Sourour Neji1, Fatma
Cheikhrouhou1, Hayet Sellami1, Stéphane Ranque2, Ali Ayadi1
1
Habib Bourguiba Hospital: Sfax - Tunisia, Tunisia; 2Laboratoire
de Parasitologie-Mycologie, Université de la Méditerranée, AP-HM
Timone, Marseille France
Nikolić P., Mehle N., Gruden K., Ravnikar M., and Dermastia M. (2010).
Molecular and Cellular Probes 24: 303-9.
Background. Improving our ability to detect Aspergillus DNA will
contribute to the diagnosis of invasive aspergillosis (IA). This study
aimed at comparing a real-time PCR assay and a PCR-ELISA
assay in both serum and bronchoalveolar lavage (BAL) samples
forthe diagnosis of IA in patients with hematological malignancies.
Patients and methods. 163 neutropenic patients at risk of IA in the
hematology wards of the Sfax University Hospital, were
prospectively studied. BAL samples, if available, and serum were
taken twice weekly. The morphological identification of Aspergillus
isolates collected from BAL samples was verified by internal
transcribed spacer 1 (ITS1), 5.8S, and ITS2 region rRNA
sequence analysis. Using a nested case-control design, both PCR
assays were performed on proven and probable IA cases and
matched control samples. Results. One proven, 31 probable, and
15 possible cases of IA were diagnosed on the basis of
EORTC/MSG criteria. The ITS sequencing proved that all isolates
collected from BAL were Aspergillus flavus. Real-time PCR, PCRELISA, and galactomannan antigen (GMA) assays performed on
459 serum samples found 93.8%, 96.9%, and 100% sensitivity,
respectively. Specificity was 100%, 100%, and 91.5%,
respectively. In 42 BAL samples, sensitivity was 64.3%, 71.4%,
and 85.7%, and specificity was 96.4%, 96.4%, and 92.9%,
respectively. Conclusions. While slightly less sensitive, the real
time-PCR assay was highly specific and considerably faster and
more workable than PCR-ELISA. Combining real-time PCR and
GMA detection in both serum and BAL samples enhances routine
laboratory IA diagnosis.
Pirc M., Ravnikar M., Tomlinson J., Dreo T. (2009). Plant Pathology, 58 (5):
872-881.
P043
Hren M., Boben J., Rotter A., Kralj P., Gruden K., Ravnikar M. (2007). Plant
Pathology 56 (5), 785-796.
P041
Is the JAK2 Exon 14 Deletion a Physiological Splice
Variant?
Influence of different one-step reverse transcription
real-time PCR reagents for no-doubt detection of
norovirus genogroup II
Paolo Catarsi, Vittorio Rosti, Laura Villani, Valentina Poletto,
Elisa Bonetti, Bergamaschi Gaetano, Barosi Giovanni
Unit of Clinical Epidemiology and Center for the Study of
Myelofibrosis, IRCCS Policlinico San Matteo Foundation, Italy
Marko Kolenc1, Nataša Toplak2, Minka Kovač2, Andrej Steyer1,
Mateja Poljšak Prijatelj1
1
University of Ljubljana, Faculty of Medicine, Institute of
Microbiology and Immunology, Slovenia; 2Omega d.o.o., Slovenia
Human noroviruses, members of Caliciviridae family are one of
the main cause of acute non-bacterial gastroenteritis in humans of
all ages. They appear in both, epidemic and sporadic cases.
Noroviruses can not be propagated in cell cultures. Thus real-time
PCR molecular methods for caliciviral detection, which represent
the basis of molecular epidemiology, have been introduced [1,2,3].
The aim of the study was to compare two different real-time PCR
reagents for detection of norovirus strains belonging to genogroup
II. The linear range of detection, sensitivity, efficiency of
amplification, comparison of detected fluorescence using two
different one-step reverse transcription real-time PCR reagents
were established. The results show that the use of a TaqMan Fast
In recent years the diagnosis and treatment of patients with
myeloproliferative disorders has focused on the JAK2 gene. The
V617F mutation changes the pseudokinase domain JH2 and
determines the constitutive activation of the protein. In a recent
publication, Ma and colleagues (Ma et al.. PLoS ONE (2010) vol. 5
(8)) identified an alternative splicing of JAK2 transcript that leads
to the deletion of the entire exon 14 (88bp). This mRNA has a stop
codon in exon 15 and is translated into a truncated protein that
has a deletion within the JH2 pseudokinase domain and complete
deletion of the kinase domain (JH1). In order to identify and
quantify the presence of this splice variant, the RNA extracted
from plasma, was reverse transcribed and amplified with primers
designed in exons 13 and 15 and analyzed using fluorescent
fragment length analysis. The alternative transcript was only
detected in patients with myeloproliferative neoplasms (MPNs)
(58% of patients positive for the V617F mutation and 33%
negative) in proportions ranging from 2% to 26% compared to the
normal transcript. We investigated this alternative splicing form in
RNA extracted from both granulocytes and total leucocytes of 6
MPNs patients (3 positive for the V617F mutation and 3 negative)
and 5 healthy controls. The amplification products (i.e. 273-bp for
the wild type and 185-bp for the splice variant) were obtained
using the same pair of primers. The products were separated by
conventional gel electrophoresis. The alternative spliced product
was present in all patients and all healthy individuals. We
hypothesized that the difference between patients and controls
groups observed in the cited work is due to a pathological
increase in the levels of a constitutive variant. To test this
hypothesis, we are developing a qPCR assay to determine the
amount of JAK2 splice variant relative to the normal transcript.
P044
Molecular Detection of Tranzschelia discolor using
qPCR
Paolo Catarsi1,3, Cristina Borghi1, Marina Laura1, Arianna
Cassetti1, Giacomo Morreale2, Andrea Allavena1
1
CRA-FSO, Research Unit for Floriculture and Ornamental
Species, Corso Inglesi 508, 18038 Sanremo, Italy; 2CRA – VIT,
Centre for Research in. Viticulture, Viale XXVIII Aprile 26, 31015,
Conegliano, Italy; 3Current address: Lab of Clinical Epidemiology,
IRCCS Policlinico San Matteo Foundation, Piazzale Golgi, 27100
Pavia, Italy
Prunus/Anemone rust is caused by Tranzschelia discolor, that has
become aggressive in Anemone coronaria cultivation in recent
years. Reliable detection of plant pathogens in propagation
material crops and ornamental plants is matter of great economic
interests because of farmer yield losses. Furthermore the supply
of of infected seed can lead to protracted litigation. Aim of our
study was to develop a qPCR technique useful to detect
Tranzschelia discolor in both seed derived rhizomes (i.e. the
propagation material of Anemone) and in epigeal tissues of the
plants. This technique can be useful for both its phytosanitary
application (i.e. the detection of the pathogen in plant nursery
facilities) and its usage as a powerful method to detect the
presence of mycelium in various tissues of the Anemone plant
during fungus migration. qPCR primers were designed on a DNA
segment coding for a ribosomal RNA. The qPCR reaction allowed
us to efficiently detect the fungus in seed derived rhizomes. This
finding is in agreement with the hypothesis that infected
propagation material accounts for yield losses in the field. qPCR
analysis confirmed the migration of the fungus in epigeal tissues of
the plant. Furthermore, qPCR data were used to set up a LAMP
based procedure, which might allow pathogen on-site detection.
P046
Multiplex Real-Time PCR for Detection of Shiga toxin
and Intimin genes of pathogenic Escherichia coli
Melanie Pavlovic, Regina Konrad, Sabine Wolf, Marion
Lindermayer, Jasmin Fräßdorf, Ulrich Busch, Ingrid Huber
Bavarian Health and Food Safety Authority, Germany
Some E. coli strains harbour specific virulence factors which
classify them as pathogens. In human they mainly cause
infections of the gastrointestinal and urinary tracts and are among
the most frequent bacterial causes of diarrhea. Especially in young
children, infections with Shiga toxin producing enterohaemorrhagic
E. coli (EHEC) can lead to the life threatening hemolytic uremic
syndrome (HUS). EHEC and enteropathogenic E. coli (EPEC)
strains can induce attaching and effacing lesions. They harbor the
locus of enterocyte effacement (LEE) pathogenicity island with the
intimin gene eae. Intimin mediates the intimate attachment of
bacteria to epithelial cells. The diagnosis of EHEC strains in stool
and food is based on detection of Shiga toxin genes and of EPEC
strains on the eae gene, respectively. We developed a multiplex
real-time PCR assay for simultaneous detection of intimin genes
and all so far known variants of Shiga toxins 1 and 2 (stx1 and
stx2) for diagnostic samples on a Lightcycler® 480 machine with
hydrolysis probes (Pavlovic et al., 2010). An internal amplification
control was included. The multiplex PCR was tested on 30 E. coli
reference strains and 174 well characterized isolates from our
routine diagnostic. 68 strains representing other gastrointestinal
pathogens, normal gastrointestinal flora, or closely related bacteria
gave no signal in the multiplex PCR. The detection limits were five
genome equivalents for stx2 and 50 genome equivalents for eae
and stx1. Before implementing the multiplex PCR in the routine
diagnostic, we compared a set of samples examined with the
newly developed method and the currently used real-time PCR.
Overall 95% of the results were identical for the stx1 and stx2
genes (n = 182 each) and 91% (n = 54) for the eae gene. In fact
we detected more stx and eae genes in our samples with the new
multiplex PCR because all variants of the genes are included.
Additional samples are currently tested.
Pavlovic M, Huber I, Skala H, Konrad R, Schmidt H, Sing A, Busch U.
Development of a multiplex real-time polymerase chain reaction for
simultaneous detection of enterohemorrhagic Escherichia coli and
enteropathogenic Escherichia coli strains. Foodborne Pathog Dis. 2010
Jul;7(7):801-8.
New qPCR Applications:
Method Optimisation & Standardisation
P045
P047
Scaling Semiconductor Sequencing from Microbial to
Human Genomes on a Single Platform
Alain Rico1, Jonathan M. Rothberg2
1
Life Technologies, France; 2Ion Torrent, USA
Ion Torrent has invented the first device—a new semiconductor
chip—capable of directly translating chemical signals into digital
information. The first application of this technology is sequencing
DNA. The device leverages decades of semiconductor technology
advances and in just a few years has brought the entire design,
fabrication and supply chain infrastructure of that industry - a
trillion dollar investment—to bear on the challenge of sequencing.
The result is Ion semiconductor sequencing, the first commercial
sequencing technology that does not use light, and as a result
delivers unprecedented speed, scalability and low cost. All of
these benefits are a result of applying a technology that is
massively scalable, as proven by Moore’s Law, to a task that has
traditionally
used
optics-based
solutions.
Optics-based
sequencing works in a linear fashion: Increasing capacity requires
increasing the number of signals that must be read, which in turn
results in longer runtimes, higher capital costs and ever more
sophisticated optics.
Natasha Paul
PCR is a widely used scientific tool whose specificity can be
increased by the use of Hot Start technologies. Although many
Hot Start technologies exist, recently developed CleanAmp™
dNTPs are a distinct approach that employs modified nucleoside
triphosphates with a thermolabile protecting group at the 3'hydroxyl. The presence of the protecting group blocks low
temperature primer extension, which can often be a significant
problem in PCR. At higher temperatures, the protecting group is
released to allow for incorporation by the DNA polymerase and
more specific amplification of the intended target. These modified
dNTPs provide comparable performance to other Hot Start
technologies and can be used with thermostable DNA
polymerases to turn a reaction into a Hot Start version. This
thermolabile chemistry can be applied to dNTP analogs such as
dUTP, which is used in UNG decontamination methods, and 7deaza-dGTP, which is used to amplify difficult GC-rich targets. In
addition, further studies have led to the development of 3'protecting groups that deprotect more quickly than the current 3'modification group, allowing these modified dNTPs to be used in
fast PCR. With the evolving chemistry of the hot start dNTPs, the
areas of application benefiting from the versatility and flexibility of
this technology continue to grow.
P048
Lyophilized Mix of Enzyme, Probes, and Primers: A
simple and highly sensitive solution for qPCR
applications.
Nanette Umblas, Chieh-Yuan Li, Lin Lin Huang, Kathy Lee,
David Keys, Kate Hames, Madhu Augustine, Stephen
Hendricks
qPCR reactions require a number of components including
dNTPs, salts, buffers, enzyme, qPCR assay primers and probes,
and sample. Numerous pipetting steps may introduce errors and
are not amenable for high throughput applications. A number of
time consuming pipeting steps can be eliminated when you use
pre-formulated primer/probe mixes such as Applied Biosystems’
TaqMan® Gene Expression Assays, with optimized enzyme
mixes. However, a single pipetting step workflow from sample to
qPCR reaction is highly desirable for new applications, especially
in clinical research and molecular diagnostics, where minimal
handling is often required. Thus, a solution that provides ease of
use, without compromising precision and accuracy is highly
advantageous. To meet this need we have developed a qPCR
solution that centers on lyophilization of probe, primers and
enzyme mix. Lyophilization, also known as freeze drying, is a
widely used technique in food and pharmaceutical industries for
room temperature storage and transportation of compounds. We
have successfully applied this technique to qPCR such that probe
and primers for any gene can be combined with enzyme into a mix
that is stable at room temperature. With the new lyophilized
format, end users only need to add their sample solution into the
reaction tube (8-well tube strips or 96-well plates) where the
lyophilized assay and enzyme mix has been pre-dispensed.
Besides providing convenience, cost savings and minimal
pipetting error, this format allows increased sample volume, thus,
potentially doubling the sensitivity of detection compared to wet
assays. Here we present the challenges of applying lyophilization
in qPCR, lyophilized assay performance data when compared to
wet assays (R^2 = 0.98; PCR efficiency ~100%), and preliminary
stability data when stored under different conditions.
P049
Extraction protocol to increase the yield of circulating
fetal DNA in maternal plasma
Lai-on Chu, C.Y. Rachel Yeung, K.F. Joseph Chow
DiagCor Bioscience Inc. Ltd., Hong Kong S.A.R. - China
Background: Compare to the current invasive prenatal diagnosis
methods,non-invasive approach is an ideal way to perform
diagnostic test in much earlier stage of pregnancy while reducing
the chance of miscarriage minimum. However, the nature of low
concentration of cell free fetal DNA (cffDNA) in maternal
circulation is the major technical challenge to achieve effective
non-invasive prenatal diagnosis. We present here a sample
collection and extraction workflow generating substantial increase
in extraction yield and total amount of fetal DNA in maternal
plasma, increasing the feasibility for clinical application of noninvasive prenatal diagnosis. Methods: 29 maternal blood samples
were collected from gestation 8-15 weeks with Streck’s Cell-free
DNA BCT tube and cffDNA were extracted by QIAamp Circulating
Nucleic Acid Kit. Cell free Fetal DNA content and ratio in plasma
were quantified and estimated by real-time PCR. Results: A total
of 29 plasmas from maternal blood (gestation week ranged from 8
to 15 weeks) were analyzed. The total DNA isolated from 5ml
plasmas ranged from 337.72 – 1243.88 GE/ml. 16 of 29 maternal
plasma samples were Y-positive, from which the Y specific
sequences were used to quantify the fetal DNA content. Fetal to
total DNA ratio in plasma ranged from 3.45% to 15.53%, whereas
the mean fetal DNA isolated was 51.80 GE/ml (ranged 23.72 –
87.15 GE/ml). The 5ml protocol can recover more than 100 GE of
fetal DNA (ranged 118.6-435.75 GE) from 10 ml of maternal
blood. Conclusions: This workflow enables cffDNA to be
preserved and collected in higher yield than normal collection
method. Compare to the literature reported, our fetal to total DNA
ratio was 2 folds better (i.e. median ranged from 9.21 and 14.06%
compared to 4.8% and 4.1% for first and second trimester
respectively). Furthermore the collected Blood sample can be
preserved up to 7 days with no significant change. Therefore, this
workflow greatly opens up the possibility of performing
downstream application for non-invasive fetal diagnosis.
P050
Quantitative detection of pathogens and of
mRNA/gene mutants: Internal and external reference
RNAs and DNAs for the development and high level
quality & performance controls of real-time RT-PCR
and PCR assays
Guido Krupp, Andreas Hanne, Peter Scheinert
AmpTec GmbH, Germany
One important aspect of using synthetic internal control RNAs:
false negative results are easily discovered. Availability of
customized RNAs allow monitoring of both steps in RT-qPCR
tests: the crucial cDNA synthesis and the subsequent PCR
amplification. High quality dilution series of positive controls allow
routine implementation of performance control of the tests and
guarantee continuous high quality standards. The development of
mutant screening tests is straightforward and any desired
sequence variant is available in homogeneous form, independent
from the limited availability of (uni-variant) biological samples. An
important aspect of pathogen detection is the evaluation of test
specificity: only the intended target sequence(s) give positive
results, closely related species are not detected and give negative
results. Availability of the corresponding sequence panels can be
challenging for new test developers without resources that have
been accumulated over many years. The availability of customized
reference sequences offers unlimited possibilities for any facility
and for new fields. Applications of these reference nucleic acids
are demonstrated with instrument platforms from the
manufacturers ABI, Qiagen and Roche. Examples are shown for
quality & performance control and for the detection of the closely
related viral sequences HSV-1 and HSV-2 in one single test.
P051
Validation of appropriate reference genes for the
normalization of real-time quantitative RT-PCR data
obtained from platelets of post-myocardial infarction
patients
Amir Houshang Shemirani1,2, Katalin Szilvia Zsóri1,2, László
Muszbek1
1
Debrecen University, Hungary; 2Sátoraljaújhely hospital, Hungary
Human blood platelets contain minute amounts of translationally
active mRNA. Genetic analysis of platelet mRNA islimited by small
yield of plateletmRNA and the risk of leukocyte contamination
duringplatelet preparation.Previous reports on platelet mRNA
expression have used conventional reference genes, but there
has been no report of studies to check for the validity of these
reference genes. In other words, reference genes had not been
thoroughly investigated in platelet. Method: We isolated leukocytedepleted platelets from ten mL blood by triple centrifugation. We
tested the reliability, leukocyte contamination, and integration of
platelet RNA by RT-qPCR to detect specific transcripts.
Spectrophotometry (NanoDrop) was used for the quality
assessment of isolated RNA. RT-qPCR was performed on
LightCycler® 480. The software NormFinder and geNorm were
used to identify the most suitable reference genes. Expression
proﬁles of 9 genes ACTB, EAR, GAPDH, HDGF, GNAS, ANAPC5,
OAZ1, EEF2 and CFL1 were established from 20 patients with
myocardial infarction. Results: Nanogram quantities of plateletspecific cDNAs were produced from 10 mL of whole blood. ACTB
and HDGF (by geNorm) and ACTB (by NormFinder) were the
most stably expressed genes in platelets of patients with the
history of myocardial infarction. Conclusion: In platelets gene
proﬁling studies of patients after myocardial infarction the genes
ACTB and HDGF in combination are recommended as reference
genes for normalization.
P052
Elisabeth Lill Andreassen, Olav Lanes, Morten Elde
ArcticZymes AS, Norway
Exogenous RNA control for indication of reaction
inhibition in the RT-qPCR workflow
PCR is sufficiently sensitive to detect single copy genes from
single cells. Unfortunately, this extreme sensitivity of PCR also
restricts the method, making it prone to false-positive results due
to contamination. One of the major sources of these false-positive
results is carry-over contamination of amplification products from
previous PCRs (1). As the amplicons are both identical to the
target molecule and produced at a very high copy number during
PCR, they constitute a serious threat, particularly to forensic,
diagnostic and palaeogenetic analysis (2). With the advent of the
uracil-N-glycosylase (UNG) de-contamination technology, this
problem became widely solved; however, reactivation of UNG
post-PCR has restricted its use (3,4,5,6). Upon reactivation of
UNG, the newly produced PCR products quickly degrade, thus
impairing downstream analysis such as cloning, sequencing and
genotyping. So the complete and irreversible inactivation of UNG
is of pivotal importance for post-PCR applications. In our recent
studies we have demonstrated how a range of commercially
available UNGs fail to meet these criteria. Residual UNG activity
was measured by incubating UNG-treated PCR products at room
temperature at different time intervals and the extent of DNA
degradation was evaluated on agarose-gels. We further
sequenced the UNG-treated PCR products and evaluated the
quality of the sequences. Our study showed that the only UNG
that became completely and irreversibly inactivated by moderate
heat-treatment, was cod UNG. The post-PCR sequencing results
showed that every UNG tested, except the cod UNG, gave poor or
unreadable sequences. Hence, by employing cod UNG as the decontamination enzyme in PCRs, a range of downstream analysis
of PCR products can be performed without the probability of UNGreactivation impairing the results.
James Covino2, Zaklina Strezoska2, Devin Leake2, Annaleen
Vermeulen2, Melissa Kelley2, Jaakko Kurkela1
1
Thermo Fisher, Finland; 2Thermo Fisher Scientific, Lafayette,
Colorado
Inhibitors of reverse transcriptase quantitative PCR (RT-qPCR)
are commonly present in experiments and not easily recognized in
experimental set-ups. The primary challenge is that methods such
as spectrophotometric techniques do not always detect RT-qPCR
inhibitors. The Thermo Scientific Solaris RNA Spike Control Kit
was designed as an exogenous control to identify the presence of
reaction inhibitors including those commonly carried through
processing steps to isolate RNA such as EDTA, phenol, heparin
and EtOH. Here we describe applications for this exogenous
control to identify the presence of reaction inhibition and illustrate
the impact inhibition can have on results and data interpretation.
Experimental results demonstrate RNA Spike Control provides
accurate identification of RT-qPCR inhibition and the ability to
predict when inhibition has been removed, for example, by dilution
of the RNA sample into the reverse transcription reaction. The
RNA Spike Control identified RT-qPCR inhibition in a gene
expression assay, explaining the variable results obtained.
Furthermore, the use of RNA Spike protocol was compared to
current inhibition identification protocols and was found to be an
easy and succinct workflow, offering a clear advantage of this
technology.
P053
Internal controls for quantification of alternatively
spliced transcripts by real-time PCR
Julia Meiser, Julia Frühwald, Stephan E. Philipp
Institute for experimental and clinical pharmacology and
toxicology, University Saarland, 66421 Homburg, Germany
More than 90% of human genes express mRNAs that undergo
alternative splicing resulting in an extensive collection of isoforms
that are required for a huge variability of functions. The ratio of the
variants is tightly controlled and needs to be determined precisely.
Real-time reverse transcription polymerase chain reaction (RTqPCR) is the method of choice to quantify mRNA expression
levels and has also been adapted to measure the ratio of splice
variants derived from a single gene (1). However, commonly used
assays do not compensate for differences in the reverse
transcription efficiencies among the variants and do not include
internal controls that reveal experimental errors. Here we present
an expanded method to quantify the ratio of alternatively spliced
transcripts by RT-qPCR. In addition to transcript specific primers
the method includes control primers that allow compensation of
variations during reverse transcription. Furthermore they serve as
an independent control of the total number of transcript. To
estimate the PCR efficiencies we used the comparative
quantitation tool of the rotorgene software which is based on the
determination of the second derivative maximum (SDM) of the
amplification curve. Comparison of efficiency corrected values to
those obtained by standard curves as well as a new algorithm to
calculate the PCR efficiencies (LinReg, (2)) showed that SDMdata were equally reliable and that standard curves are
dispensable. The one-step protocol can be easily adapted to most
splice events and allows simultaneous identification of two
distinguishable products specific for both transcripts. We show
that the method is applicable to a broad range of template
concentrations. Furthermore, it is possible to determine minute
changes of the ratio of two variants of ~ 10 %.
1. Vandenbroucke, I. I., Vandesompele, J., Paepe, A. D., and Messiaen, L.
(2001) Nucleic Acids Res. 29, E68
2. Ruijter, J. M., Ramakers, C., Hoogaars, W. M., Karlen, Y., Bakker, O.,
van den Hoff, M. J., and Moorman, A. F. (2009) Nucleic Acids Res. 37, e45
P054
Contamination control with the heat-labile cod UNG
ensures accurate and reliable results in your post-PCR
analysis
1. Kwok, S., Higuchi, R. (1989); Nature 339:237–238
2. Pruvost, M.,
Grange, T., Geigl, E.M. (2005): BioTechniques 38:569–575
3. Longo,
M.C., Berninger, M. S., Hartley, J. L. (1990); Gene 83:125–128 4. Sobek
H., Schmidt M., Frey B., Kaluza K. (1996); FEBS Lett 388:1-4 5. Thornton,
C. G., Hartley, J. L., Rashtchian, A. (1992); Biotechniques 13(2):180–183
6. Champlot, S. et al (2010) PLoS ONE 5(9):e13042
P055
NOVEL APPROACHES IN MANUFACTURING HIGH
QUALITY REAGENTS FOR IN-HOUSE PCR
DIAGNOSTICS
Muriel Craynest
EUROGENTEC, Belgium
Background. Emerging infectious diseases like H1N1, have
prompted the need for rapid development of routine PCR testing
by service laboratories. The rapid need for such tests requires inhouse development and manufacturing.
However, like
commercial kits, in-house PCR needs to be built with reagentsof
high and consistent quality to ensure reliable and accurate assay
performance. Currently, most laboratories will source ‘researchgrade’ oligos (produced under ISO 9001 quality system) rather
than “diagnostic” GMP reagents(produced under ISO 13485) to
save costs. From a quality perspective this is farfromoptimal.
Moreover, with more and more medical labs upgrading their
quality system to ISO 15189, control measures over critical
reagents and their suppliers must be implemented. As oligos are
the most critical components of PCR, an innovative manufacturing
solution is proposed in this paper: Clinical Lab Oligos. Methods.
FMEA-based risk analysis of the oligo production process was
performed, followed by appropriate measures to minimize risks for
diagnostic users. Risks were classified as low/medium/high, based
on Severity (impact for customer), Occurrence & Detection.
Results. We identified 471 risks in the first round. Using a
reiterative process, measures were implemented to reduce all
risks to “low” classification. Important measures included, the use
of classified clean rooms to eliminate contamination risks, GMP
documentation
(SOP
and
checklists),
and
validated
instrumentation and QC methods. The improved production
process is managed according to ISO 13485. Conclusion. Clinical
lab oligos will significantly add to further quality improvement of inhouse PCR Diagnostics.
qPCR Biostatistics & Bioinformatics
P056
Real-time PCR quantifications in presence of inhibitors
using SOD and Cy0 methods.
Davide Sisti, Michele Guescini, Renato Panebianco, Pasquale
Tibollo, Michela Mantuano, Marco Rocchi, Vilberto Stocchi
University of Urbino, Italy
Introduction: Real-time PCR has become the technique of choice
for absolute and relative nucleic acid quantification. The gold
standard quantification method in real-time PCR (Ct) assumes that
the compared samples have similar PCR efficiency. Recently, the
real-time PCR basic quality control guidelines of quantification
have been proposed, in order to achieve a reliable quantification.
However, in some biological samples, some inhibitors can be
copurified with DNA template and often it is hard to remove them.
Consequentially, differential inhibition could be observed resulting
in real-time PCR quantification bias. In order to avoid
quantification inaccuracy, due to differences in amplification
efficiency among run samples to be compared, outlier detection
methods, based on amplification estimation, have been reported.
Recently a new method, namely SOD, showed a high sensitivity
and specificity to detect outlier runs. SOD (shape outlier detection)
compares shape of amplification curve of experimental runs with
standard curve, without comparing efficiency. Moreover, a new
DNA quantification method, called Cy0, show better accuracy in
presence of inhibitors. Hence, we compared DNA quantification
obtained using Cy0 and Ct methods, considering SOD value of
each run. Material and methods: The DNA consisted of a pGEM-T
(Promega) plasmid containing a 104 bp fragment of the
mitochondrial gene NADH dehydrogenase 1 (MT-ND1) as insert.
Real time PCR amplifications were conducted using LightCycler®
480 SYBR Green I Master (Roche) according to the
manufacturer’s instructions. Ct (fit point method) was determined
by the LightCycler® 480 software version 1.2. The Cy0 value is
the intersection point between the abscissa axis and tangent of
the inflection point of the Richards curve obtained by the nonlinear regression of raw data. Shape based kinetic outlier
detection (SOD) was based on the shapes of the amplification
curves. SOD value is based on Mahalanobis distance; it is a
useful way of determining the similarity of an unknown multivariate
sample set to a known one, with multinormal distribution. Results:
For each runs, in presence of biological inhibitors such as tannic
acid (range: 0.39– 100 mg/mL ), IgG (range: 0.39– 200 mg/mL ) or
quercitin (range: 0.31– 40 mg/mL ), Cy0 and Ct values have been
calculated. When estimated DNA concentrations, using Ct
method, were significantly different from expected values, due to
inhibitor presence, SOD efficiently marked these amplification
profiles as outliers. Instead, DNA quantifications obtained with Cy0
method, were not significantly different from the expected values,
obtained from residual distribution of curve calibration runs.
Conclusion: Our results demonstrated that in presence of three
different type of inhibitors at different concentrations, SOD
efficiently detected outlier runs and Cy0 method achieved
quantifications with accuracy significantly higher than Ct method.
Web site: http://www.cy0team.uniurb.it
P057
Kinetic models of qPCR as applied to the problem of
low copy numbers
Alexander M Chagovetz, James P Keener
University of Utah, United States of America
We propose an alternative way of analyzing qPCR data based on
kinetic modelling of multiple competing reactions. Three different
models are developed for kinetically distinct cases: SYBR greentype detection, hybridization probes, and hydrolysis (TaqMan)type probes. Instead of analyzing local features of the growth
curve (Ct) we propose a global fit of the growth curve as defined
by the model. Our approach demonstrate certain advantages in
quantification of low copy number targets, which is espacially
relevant for single cell qPCR.
Proposed method allows to:
1. Estimate changes in amplification efficiency during the course
of reaction as a function of initial target concentration.
2. Expand the dynamic range of quantitations and improve its
stability
3. Reliably distinguish positive (non-zero copy number) and
negative (null) samples
P058
Modeling Immuno-qPCR to Estimate Protein Quantity
Harrison Leong, Elana Swartzman, Jennifer Holmdahl
Obtaining relative quantification information from immuno-qPCR
assays involves measuring Ct values for serial dilutions of
unknown and reference samples. In general, these curves have
log-linear ranges and, within these ranges, the curves are nonparallel. A theoretical model is developed and used to provide a
means to estimate relative quantity in a way that accounts for
these characteristics. The theory makes it clear that the method
must be calibrated and suggests several alternatives to achieve
that. Experiments have been devised and executed to generate
data with “known” underlying fold change values. A calibration
and fold change estimation approach are cross-validated against
these data. Results suggest that the method can determine fold
change within a half log.
P059
Development of a GMO screening database with
combinatory algorithms and its application in official
food and feed control
Lars Gerdes, Sven Pecoraro
The development and cultivation of genetically modified crops is
increasing globally [1]. Food and feed imports from outside the EU
will subsequently require more effort from the responsible
authorities in monitoring compliance with effective labelling
directives in the future. Our aim was the development of a
combinatory screening system to support an efficient and
comprehensive routine laboratory analysis in Bavaria, Germany.
The project consists of two parts: the development of a database
for collection and interpretation of information related to genetically
modified organisms (GMO) and the design of ready-to-use
microtitre plates (modules) for experimental screening of samples.
Different genetic elements (e.g. promoters, terminators, structural
genes) are artificially introduced into plants to establish new
genetic modifications. The introduced elements may vary between
different GMO lines, depending on the intended traits (e.g.
resistance to herbicides or insects, changes in metabolite
patterns, etc.). Screening for such inserted elements with (realtime) PCR is a common first step to analyse samples for the
presence of any genetical modification. From the pattern of
detectable and not detectable elements, valuable conclusions
about the identity of putative present GMO line(s) can be drawn.
Information about selected genetic elements from the literature [for
example 2], applications for authorisation and other (web) sources
were systematically integrated in a matrix-format. Special care
was taken to additionally record the sources of the information,
thus facilitating evaluation of screening results, and tracing of
possible errors in the matrix. Data from the matrix was accessed
with several implemented algorithms, for example to interprete the
outcome of a screening for genetic elements with the ready-to-use
microtitre modules, and thus narrow down the candidates for
subsequent identification reactions. Database and screening
modules are open for implementation of further GMO lines and
additional genetic elements. Together, data processing and
corresponding ready-to-use real-time PCR modules will support
the efficient analytical food and feed control at the Bavarian Health
and Food Safety Authority (LGL).
1. James, C., Global Status of Commercialized Biotech/GM Crops: 2009.
ISAAA Brief, 2010. 41. 2. Waiblinger, H.-U., B. Boernsen, and K. Pietsch,
Screening-Tabelle für den Nachweis zugelassener und nicht zugelassener
gentechnisch veränderter Pflanzen. Deutsche Lebensmittel-Rundschau,
2008. 104(6): 261-4.
P060
Advanced copy number variant analysis with
qbasePLUS
Biogazelle, Zwijnaarde, Belgium
Copy number changes are known to be involved in numerous
genetic disorders. Moreover, copy number polymorphisms of
various sizes are thought to contribute to normal phenotypic
variation and susceptibility to multifactorial disease. In this context,
qPCR-based copy number screening may serve as the method of
choice for targeted copy number screening as it has many
advantages over alternative methods, such as its low consumable
and instrumentation costs, fast turnaround and assay
development time, high sensitivity and open format (independent
of a single supplier). Here, we present several experiments in
which we performed targeted deletion screening in patients with
human genetic disorders. Accurate copy number calling and
objective interpretation was performed with an advanced module
for copy number analysis integrated within qbasePLUS 2.0. The
software allows the selection of more than one reference sample
for accurate copy number calling. In addition, it provides flexibility
with regard to the reference samples as these samples may have
varying copy numbers. The identified copy numbers changes are
visualized on a per sample basis and conditional bar colouring is
applied for easy detection of deletions and amplifications. In
summary, we provide guidelines for qPCR-based copy number
screening and subsequent data-analysis to improve the quality
and reliability of your results.
P061
Full-Process-Kinetics PCR analysis: a holistic model
to PCR data interpretation.
Antoon Lievens1, Stefan Van Aelst2, Marc Van den Bulcke1,
Els Goetghebeur2
1
Institute of Public Health, Belgium; 2Ghent University, Belgium
Real-time PCR analysis has become a key application in
molecular biology. Yet, despite its general use and marked
improvements in thermocycler hardware over the past decade,
little has changed in the way real time PCR data are typically
analyzed. Most current approaches assume constant efficiency
over a single reaction. Such methodology performs well as long as
long as PCR efficiency does not significantly vary between
reactions. Yet, small inaccuracies in reaction efficiency can lead to
large quantification errors. Especially when working with biological
samples, the possible presence of coextracted inhibitors poses a
challenge to data analysis. A new approach to modeling real-time
PCR data is presented, called Full Process Kinetics PCR (FPKPCR). It combines a kinetically more realistic model with flexible
adaptation to real time PCR data. The approach aims at
reconstructing the entire chain of cycle efficiencies, rather than
just the data in a limited 'window of application'. As a result, one
extracts additional information and loses a level of arbitrariness.
The method reveals the amplification steps underlying
fluorescence increases. This opens access to detailed information
about the internal efficiency behavior and derived parameters of
interest. The novel approach was tested on real time SYBRgreen
PCR data originating generated with genetically modified roundup
ready soybean (event GTS 40-3-2) genomic DNA. Addition of a
known PCR inhibitor, isopropanol, allowed to evaluate the
methods ability to distinguish inhibited from non inhibited
reactions. Results: Efficiency estimates returned by the model are
comparable in both accuracy and precision to the golden standard
of serial dilution on the one hand, and to other single reaction
efficiency calculation methods on the other hand. The changes in
efficiency throughout the reaction as described by the FPK-PCR
procedure are however considerably closer to the actual data than
other models available through the literature. The FPK-PCR
approach further allows to identify inhibited reactions by correctly
estimating maximal reaction efficiency.The approach thus
demonstrates that by implementing a global efficiency model one
can avoid the selection of a window of application. When
investigating gene expression or quantifying sample content,
differences in efficiency between samples before and after
treatment, or between a sample and a standard, introduce bias
that may skew the conclusions of an assay. FPK-PCR now allows
to avoid such bias by yielding efficiencies that are
comparablebetween reactions. Knowledge of the individual
reaction efficiencies may be used to compensate for efficiency
differences between reactions or to simply remove reactions with
aberrant efficiency from analysis. Finally, the individual cycle
efficiencies enable reconstruction of the fluorescence data,
providing a quality control over the efficiency estimate.
High Throughput qPCR & digital PCR
Next Genartion Sequencing
P062
Identification of Tumor Cell Lines by Molecular
Signature using Digital PCR and TaqMan® Copy
Number Assays
Julie Kuykendall
Digital PCR (Polymerase Chain Reaction) is an advanced
utilization of PCR technology where the number of nucleic acid
template is accurately counted. The digital PCR technology relies
on segregation of individual target template into different
reactions. This can be achieved by diluting the nucleic acid
template to the Poisson limit where in each PCR reaction contains
either a single copy of template or none. Then PCR amplification
is performed where the reaction wells contains template will show
amplification signal while the wells without template shows no
signal. The 1 and 0 scoring of the wells gives the digital nature of
this PCR practice. The positive wells are counted and the number
can be extrapolated to exact starting number of template. To
achieve statistically accurate counting by digital PCR the number
of replicates needs to be quite large. Thousands of replicates are
necessary to achieve greater than 95% confidence in the counted
number. Chromosomal rearrangement is a common feature in
tumor derived cell lines. Here, we describe an approach using
digital PCR to detect chromosomal aberration in tumor cell lines.
We use 48 TaqMan® Copy Number Assays to count the copy
number of chromosomes. Separately, we use the TaqMan® assay
targeting the RNaseP gene on chromosome 14 as a copy control.
We counted genomic DNA extracted from 6 different human
cancer cell lines (A431- epidermoid carcinoma, HeLa- cervical
adenocarcinoma, Raj- B cell lymphoma, K562- chronic
myelogenous leukemia, Jurkat- acute T cell leukemia, MCF7breast adenocarcinoma). We also used genomic DNA normal
human cell lines (one each female and male) as controls. We
found chromosome numerical aberration throughout the entire
genome for all the human cancer cell lines. Some chromosomes
are duplicated entirely where all 4 copy number assays show the
same count. While some chromosome are missing entirely where
there were no signal from all 4 assays targeting the same
chromosome. The results are consistent with the aneuploidy
nature of these tumor derived cell lines. There are also sectional
duplication and deletions within chromosome where the 4 assays
targeting the same chromosome show different counts. The
combination of chromosome numerical aberration and intra
chromosome rearrangement pattern is unique to each cell line.
This allows the identification of cell line by digital PCR signature.
The utilization of digital PCR for nucleic acid template counting
enables accurate determination of starting template. Furthermore
the absolute numerical result of digital PCR counting method
allows direct comparison between different results obtained from
different run or instruments. This is an advantage to threshold
calculated result from traditional qPCR where within-run
normalizers are required to compare between runs and
instruments. The use of digital PCR is a significant evolutionary
step in nucleic acid quantification method.
P063
P065
High throughput microfluidic dynamic array profiling
demonstrates differential gene expression of immune
related genes during early pregnancy in cattle
Parallel Preparation of Targeted Resequencing
Libraries from 480 Genomic Regions Using Multiplex
PCR on the Access Array™ System
Anna E Groebner1, Katy Schulke1, Horst D Reichenbach2,
Heinrich HD Meyer1, Susanne E Ulbrich1
1
Physiology Weihenstephan, Technische Universitaet Muenchen,
Germany; 2Bavarian State Research Center for Agriculture,
Institute of Animal Breeding, Grub, Germany
Fiona Kaper1, Jun Wang, Megan J. Anderson, Peilin Chen,
Min Min Lin, Martin Pieprzyk, Robert C. Jones, Andrew P. May
Fluidigm Corporation, 7000 Shoreline Court, Suite 100, South San
Francisco, CA 94080
Due to the genetic disparity of the semi-allogenic embryo and the
mother, potential tolerance inducing genes are required for the
establishment of pregnancy. To determine the transcript
abundance of immune relevant genes in estrus synchronized
pregnant and non-pregnant Simmental heifers, total RNA was
extracted and reverse transcribed from ipsilateral endometrial
tissue (days 12, 15 and 18 after onset of estrus) and preimplantation conceptuses (days 15 and 18). The high throughput
gene expression platform (Fluidigm® 96.96 Dynamic Array) was
used based on microfluidic dynamic arrays allowing 9´216 real
time PCR gene expression measurements per chip. The mRNA
expression of 32 genes in 94 preamplified endometrial and
embryonic samples each was measured with three replicates on a
single chip. We obtained an average standard deviation of 0.16
and a chip to chip correlation of >0.99 for all three replicates. The
platform thus allows a rapid large scale analysis of real time PCR
assays with a high dynamic range and sensitivity. Proinflammatory
cytokines analyzed were not differentially expressed. However, a
significant 18-fold (p<0.001) higher transcript abundances for
indoleamine 2,3-dioxygenase 1 (IDO1) in the endometrium of
pregnant vs. non-pregnant animals was detected. IDO1 encodes
for the initial L-tryptophan degrading enzyme of the kynurenine
pathway and is known to inhibit activity of lymphocytes.
Furthermore, β2-microglobulin (B2M), the small subunit of MHC
class I molecules was only marginally expressed on conceptus
tissue when compared to the endometrium, thus the conceptus
might avoid immunological detection by the maternal immune
system. In conclusion, our data point towards an interaction of
various tolerance inducing strategies at the embryo-maternal
interface required for the establishment of pregnancy.
Supported by DFG (Ul 350/1-2, FOR 478)
P064
RealTime ready qPCR Assays - Workflow and an
interim census of 10k+ functionally tested qPCR
assays
Christine Bruns, Manuel Dietrich, Ursula Grepl, Rita Haerteis,
Ingrid Hoffmann, Regina Huber, Bastian Kramer, Irene
Labaere, Ralf Mauritz, Elke Stadlbauer, Heiko Walch
Roche Applied Science, Germany
On the RealTime ready (RTr) configuration portal we offer pretested human, mouse and rat qPCR assays based on the unique
Universal ProbeLibrary technology. The main advantage of RTr
qPCR assays is that each individual assay is functional tested for
specificity, PCR efficiency, signal intensity etc. according to
stringent criteria and in general consistency with the recently
published MIQE guidelines. Here we provide a detailed overview
of the involved processes for the RTr on-demand qPCR assay
development. The first step involves using the comprehensive
search functionalities provided within the RTr Configurator to
identify the target gene of choice from one of the three organisms.
The in-house development workflow starts by defining appropriate
transcripts for individual targets, continues with design, testing and
assay selection procedures and concludes with the final assay
production and shipment either as single liquid assays or as
multiple dried-down assays in 96/384 well plates. All individual
process steps are described, summarizing the general
specifications as well as the experience gathered from 10k+
successfully developed qPCR assays.
Next generation sequencing platforms have dramatically reduced
sequencing costs. However, it currently remains too expensive to
routinely resequence entire human genomes in order to discover
genetic variants or somatic mutations underlying tumorigenesis.
Therefore, a need exists for multiplexed, targeted amplification
methods that allow for the analysis of multiple genomic regions in
large cohorts. Available targeted enrichment technologies are
either aimed at the capture of regions of interest from a single
sample, exhibit uneven representation or require significant
amounts of input material. The novel microfluidic platform, the
Access Array™ system, combines 48 samples with 48 primer sets
resulting in 2,304 simultaneously occurring PCR amplifications
requiring as little as 50ng DNA per sample. PCR products
generated on the Access Array™ system can be used for
sequencing on all next-generation sequencing platforms, including
454 GSFLX and Illumina GAII. To increase coverage and
throughput, PCR reactions can be multiplexed within Access Array
IFCs generating up to 480 amplicons per sample.
P066
Integration of a novel real-time PCR detection system
onto Luminex beads in a PCR reaction.
Ross Thomas Barnard1, Richard Lai1, Simon Corrie2, CheCheng Yeh1, Graeme Barnett3, John Feros3
1
School of Chemistry and Molecular Biosciences,The University of
Queensland, Australia; 2Australian Institute for Bioengineering and
Nanotechnology, The University of Queensland; 3BioChip
Innovations Pty. Ltd., Suite 4, 1407 Logan Rd, Mt Gravatt, Qld
4122, Australia
We previously reported the development of a new real-time PCR
detection system, (PrimRgloTM), that utilises “tagged” PCR
primers,
a
fluorophore
labelled
‘universal’
detection
oligonucleotide and a complementary quenching oligonucleotide
(1,2). In that solution phase embodiment, the fluorescence signal
decreased as PCR product accumulated due to the increase in
detection/quencher hybrid formed as the tagged primer is
consumed. We used influenza A matrix gene and porA and ctrA
genes of Neisseria meningitidis as targets for developing that
system. Ct values were generated and the sensitivity of the
PrimRglo system compared very favorably with the SYBR green
and TaqmanTM detection systems (1). In a new development, the
PrimRgloTM system has now been integrated with LuminexTM
microbeads, utilizing a universal capture oligonucleotide coupled
to the microbead, and a Cy3 label on the universal detection
oligonucleotide. PCR (target: influenza A matrix gene plasmid)
was carried out, with the oligonucleotide-conjugated Luminex
beads in the PCR tube throughout the PCR reaction. The PCR
reaction mix was injected into the Luminex machine at the end of
the reaction. Reactions were sampled at different cycle numbers
and the Cy3 fluorescence signal associated with the microbeads
increased in proportion to the amount of PCR reaction product.
The Luminex machine currently has the capacity to measure 96
different reaction products attached to optically distinguishable
bead sets, so the integration of the PrimRglo real-time detection
system onto Luminex beads has the potential to enable high
degree multiplexing of real-time PCR reactions, beyond that which
is currently possible with standard methods.
1 . R. Barnard, R. Lai, D. Pearson, Z.Y. Phua, D. Whiley, T. Sloots and G.
Barnett. A new multiplexable, quantitative, real-time system for detection of
nucleic acids. New Biotechnology, Volume 25, Supplement 1, September
2009, Pages S17-S18, abstracts of the 14th European Congress on
Biotechnology, Barcelona, Spain 13–16 September, 2009.
2. Barnard, R.T. & Barnett, G.R. (2007) A method and kit for analyzing a
target nucleic acid sequence. WO 2007/003017.
P067
Performance and Accuracy Breakthroughs with the
5500 Series SOLiD™ Sequencers
Oliver Stephan1, Timothy Burcham1, Patrick Kinney1, Steve
Boege1, Jon Hoshizaki1, Matt Chan1, David Cox1, Jason
Briggs1, Lee Jones1, Janet Ziegle1, Larry Joe1, Sylvia Chang1,
Kathleen Perry1, Takuya Matsui2, Shuhei Yamamoto2, Ryoji
Inaba2
1
Lifetechnologies, Inc; 2Hitachi High Technologies, Hitachinakashi, Ibaraki-ken, Japan
The 5500 Series SOLiD System incorporates a number of
improvements in system design and chemistry to give the highest
and most accurate throughput of any next generation sequencer.
The 5500 series uses a 6 lane FlowChip™ where clonallyamplified beads are deposited. The 5500xl supports two
FlowChips while the 5500 supports one FlowChip. Each FlowChip
lane is independently addressed with a proprietary, direct-injection
fluidics system which decreases run time, minimizes dead-volume
and greatly simplifies the design. Each lane can contain a distinct
sample: The system will deliver the required reagents to each
lane, independent of the other lanes, using only the reagents
required. Imaging time on the 5500 is made faster by
incorporating an innovative fast filter wheel, hardware autofocus,
and fast camera. Images are processed in real time using a
workstation-class computer utilizing state-of-the-art algorithms and
GPGPU. The amount of data generated and saved is reduced by
an order of magnitude. Chemistry improvements include longer 75
bp reads (forward), 35 bp (reverse paired end), and 2x60 long
mate pair. Accuracy improvements using the Exact Call
Chemistry™ system increase accuracy to 99.99% allowing
heterogeneous SNP detection at levels requiring much less
redundancy and improved confidence levels. Users can expect
data rates of 20-30 Gb/day with the 5500xl. Results from several
library types and applications will be shown, including run times.
Pre-release results using Nanobeads will also be shown with data
rates >40 Gb/day and typical throughput of greater than 350
Gb/run.
The transcriptome represents a comprehensive set of transcribed
regions throughout the genome. Studying transcriptome dynamics
provides important insights into the functional elements of the
genome, their expression patterns and the regulation of
transcribed regions. Recently, Next Generation Sequencing
methodologies have been introduced into transcriptome research,
with mRNAseq currently being the most innovative and highthroughput method for expression profiling. Next to comparative
gene expression analyses, mRNAseq can be used for discovering
the full extent of 5' and 3' untranslated regions, novel splice
junctions, novel transcripts, alternate transcription start sites, and
rare transcripts. The goal of our research project is to apply
mRNAseq to study the transcriptome of rice roots when
challenged by nematode infection. Rice has been intensively
studied in the past decades, since it is one of the most important
crop plants worldwide and an excellent model system for
monocotyledonous plants. The rice root knot nematode
Meloidogyne graminicola and the migratory root rot nematode
Hirschmanniella oryzae are two agronomically important rice
pathogens with very different lifecycles. Even though they cause
substantial harvest losses in rice growing areas throughout the
world, almost no fundamental research has been performed on
these nematodes and their interaction with the rice plant.
Comparing the transcriptome in rice upon infection with these
pathogens will provide important insights into general and specific
defence strategies of the rice plant towards nematodes. To test
the robustness of mRNAseq for identifying differential expression
in plant tissue, the transcriptome of uninfected rice roots and root
tips was first evaluated. On average 20 million reads per sample
were generated (76 nt reads, single end), they were mapped onto
the rice genome and differential expression was evaluated using
Bayseq. Our analysis uncovered over 2000 DEGs (differentially
expressed genes), of which 1800 showed higher expression in the
roots, whereas 390 are overexpressed in the root tips (at an FDR
cut-off of 0.05). A subset of these DEGs were independently
validated using qRT-PCR analysis, all of which confirmed the
differential expression pattern in rice roots and tips obtained by
sequencing.
P068
qPCR in immune system monitoring : a high
throughput method
Maxime Lilian Dooms, Flore Depeint, Afif M Abdel Nour
The impact of food consumption on the immune system is fast
becoming a major target for human nutrition, especially in the area
of health claims. When developing methods for application in
clinical trials, major concerns are cost effectiveness and subject
well being. Based on these criteria, we developed a method using
RT-qPCR leading to repeatable data in less than 48 hours. Human
whole blood is collected during the dietary intervention trial and
challenged in vitro with lipopolysaccharide (LPS), resulting in an
inflammatory reaction and a shift in expression of immune
markers. This shift can be measured using RT-qPCR performed
on RNA extracted after incubation. Relative expression of the
target gene for each subject is normalized against a set of
reference genes. Solaris (Thermo Scientific) specific primer and
probes targeted to genes of interest, such as cytokines TNF alpha
or IL6 are used to quantify genes expression. Those primers and
probes combinations target shorter areas than usual, allowing a
wider choice in amplicon location, while keeping melting
temperature constant. The use of one-step protocols, ready to use
optimised mixes including primers and probes, reduces the time
required for setting up a qPCR as well as risk for handling failures.
P069
Transcriptome analyses in rice: validation of
mRNAseq using qRT-PCR
Tina Kyndt1, Geert Trooskens1, Simon Denil1, Tim Demeyer1,
Annelies Haegeman1, Wim Van Criekinge1,2, Godelieve
Gheysen1
1
Department of Molecular Biotechnology, UGent, Belgium;
2
NXTGNT, UGent, Belgium
Single-cell qPCR
qPCR from limited material
P070
At the single cell level cellular noise challenges
normalization
Richard Fekete, Ron Abruzesse, Elena Grigorenko
As expected, biological systems such as tissues exhibit
considerable phenotypic variation. Surprisingly, variation even
occurs in “clonal” populations. Until now this heterogeneity has
been attributed to differences in cell cycle, microenvironments, or
cell type. These differences would be expected to affect genes
within a cell in a similar manner, i.e. cells at the same point in the
cell cycle would have the same gene profile. However, it has now
been shown that there are also differences within identical
biological processes and this has been referred to as “noise”.
These differences likely result from stochastic events within a cell
due to the limited numbers of components such as RNA
polymerases. To look at the effect of noise on gene expression in
a large number of genes we used the Cell Sensor AP-1-bla ME180 cell line as a model system. We treated cells with epidermal
growth factor to stimulate the RAS and MAPK pathways and
chose target genes predicted to be up- and down-regulated. In
order to analyze gene expression profiles from a large number of
individual cells we used the Single Cell-to-Ct kit. This kit includes
optimized reagents for sample preparation, reverse transcription,
preamplification and qPCR. We compared normalized expression
levels of a number of target genes (normalized to a variety of
control genes), with non-normalized expression levels. We then
compared this data to samples of cells prepared en masse. We
found that for samples prepared en masse, gene expression
levels varied from sample to sample. However, expression levels
of all genes changed equally between samples. This allowed
sample to sample differences to be eliminated when target genes
were normalized to control genes. When analyzing single cells we
also found that expression levels of genes varied from cell to cell,
however, in contrast to cells analyzed en masse, expression of all
genes did not change equally from cell to cell. This caused the
normalized expression profiles to vary widely from cell to cell, and
change depending on which genes were used to normalize. By
using a digital approach to analyze RNA expression profiles we
demonstrate that gene expression from single cells is affected by
many factors that are not relevant to the analogue way of
analyzing cells en masse. This study challenges the well
understood field of gene expression, which routinely uses control
genes to normalize expression profiles and demonstrates that
understanding expression profiles from single cells will require a
different method of analysis and presentation.
P071
Two different astrocytes subpopulation in cortex of
mice
Vendula Rusnakova1, Pavel Honsa2, Miroslava Anděrová2,
Mikael Kubista1
1
Institure of Biotechnology, Czech Republic; 2Isntitute of
Experimental Medicine
We classified two astrocytic populations in the cortex of
GFAP/EGFP mice, high response and low response astrocytes,
which differ in the extent of cell swelling during the 20 minute
oxygen-glucose deprivation. Based on the pharmacological study
we hypothesize that the existence of two astrocytic population in
the cortex of GFAP/EGFP mice might result from a distinct activity
or expression pattern of excitatory amino acid transporters
(EAATs), Na+, K+, Cl- cotransporters, volume regulated anion
channels or two pore potassium channels. We performed gene
expression profiling of 180 single cell astrocytes to see different
groups, 8 different genes were analysed. We found two different
groups of cells by using three independent different statistical
approaches (Principal component analysis, hierarchical clustering,
Kohonen self organizing map). We identified two subpopulations
of astrocytes with distinct gene expression profiles.
P072
Gene expression analysis of formalin-fixed paraffin
embedded laser microdissected endometrial tissue
sections
Anna E Groebner1, Nevena Kirova1, Harald Welter2, Artur
Mayerhofer2, Heinrich HD Meyer1, Susanne Ulbrich1
1
Physiologie Weihenstephan, Technische Universitaet Muenchen,
Germany; 2Institute for cell biology, Ludwig Maximilian Universitaet
Muenchen, Germany
Establishment of pregnancy is dependent on an accurate maternal
reaction towards the ruminant pregnancy recognition signal
interferon-tau (IFNT). To determine cell type specific responses of
interferon
stimulated
genes,
mRNA
expression
of
myxovirusresistance 2 (Mx2) and indoleamine 2,3-dioxygenase
(IDO) was analyzed in microdissected formalin-fixed, paraffinembedded (FFPE) endometrial samples from pregnant and nonpregnant heifers. One mm2 of luminal (LE) and glandular
epithelium (GE) of the Zona functionalis (ZF) and -basalis (ZB), as
well as stroma of ZF and ZB from bovine endometrium (n=3,
respectively) were collected via laser microdissection (LMD, Laser
Microbeam system, P.A.L.M. Microlaser Technologies). Total RNA
was extracted (RNeasy FFPE, Qiagen), reverse transcribed
(MMLV reverse transcriptase, Promega) and preamplified
(TaqMan® PreAmp Kit, AppliedBiosystems). Appropriate
reference genes for normalization were chosen using GenEx Pro
Ver 4.3.4 (multiD Analyses AB). In pregnant animals, high
transcript abundance was detected for the interferon stimulated
gene Mx2 in every cell type analyzed, showing a mean of
5.3 ± 1.4 ΔCq ± SEM in pregnant vs. 1.9 ± 0.6 ΔCq in nonpregnant endometrium. Gene expression for IDO increased
likewise 11-fold (-0.5 ± 1.4 vs. 2.99±1.4 ΔCq). Interferon
regulatory factor 2 (IRF2) expression differed neither in LE (mean
-0.85 ± 0.34 ΔCq) nor GE (ZF) (mean 0.001 ± 0.19 ΔCq) with
respect to the status. On the contrary, a differential spatial
expression pattern with higher abundance in stroma cells (ZF) of
pregnant animals (-1.4 ± 0.8 ΔCq) than in non-pregnant animals
(0.8 ± 0.13 ΔCq) was observed for IRF2. This is in contrast to the
pregnant ewe, were expression of Mx2 increases primarily in the
LE and superficial GE (ZF), while the IRF2 is not expressed in
stroma cells. Thus, gene expression analysis on FFPE LMD tissue
sections points to species specific spatial differences in gene
expression during preimplantation.
Supported by DFG (Ul 350/1-2, FOR 478)
P073
Assessing heterogeneity at a single cell level in a
clonal population of transformed epidermal
keratinocytes
RM Teisanu1, H Richter2, J Moore3, A Paillusson2, O
Haglebüchle2, J Weber2, K Harshman2, Y Barrandon1
1
Laboratoire de Dynamique des Cellule Souches, Institute of
Bioengeneering, Ecole Polytechnique Fédérale de Lausanne;
2
Lausanne Genomics Technologies Facility (GTF), Center for
Integrative Genomics, Université de Lausanne; 3Fluidigm
Corporation, South San Francisco, CA USA
Human epidermal keratinocytes can be extensively cultured in
vitro, resulting in the formation of epithelial colonies maintained by
a stem cell hierarchy similar to that of normal epidermis. Due to
technical limitations, efforts to determine the molecular identity of
epidermal stem cells and their progeny generally rely on
assessing gene expression levels in large populations of cells.
The major caveat of this approach is the averaging of gene
expression in a functionally non-homogenous population of cells,
thus preventing the identification of individual signatures. The
purpose of this study was to optimize methods that will be further
employed in determining gene expression signatures of epidermal
stem cells and to determine the level of heterogeneity in a clonal
population of immortalized human epidermal keratinocytes (GMA
24). The expression level of 32 genes involved in proliferation,
differentiation and stemness maintenance was determined in
individual GMA24 cells bearing a fluorescent reporter of the S, G2
and M phases of the cell cycle (the fluorescence ubiquitination cell
cycle indicator (FUCCI)). To this end we employed a novel
technology based on microfluidics chips (Fluidigm’s BioMark
system) that allows the assessment of 96 genes in 96 individual
samples. Data analysis allowed the classification of genes
according to their variability from cell to cell in low, intermediate
and high variability genes. Non-expressed genes were not
included in the analysis. According to this classification, stable
genes represented 30.4% of the expressed genes. Importantly,
56.5% belonged to the highly variable genes, demonstrating that
despite the clonal origin of these cells, considerable gene
expression variability exists at single cell level. Further studies are
required to assess whether this heterogeneity results in functional
differences between cells. In conclusion, novel technology
enabling the simultaneous analysis and comparison of gene
expression in single cells reveals a previously unrecognized
heterogeneity in a clonally derived population of cells.
P074
Gene Expression Profiling of Duodenal Biopsies
Discriminates Celiac Disease Mucosa from Normal
Mucosa
Hanna Bragde1, Ulf Jansson2, Ingvar Jarlsfelt1, Jan
Söderman1
1
Division of Medical Diagnostics, Ryhov County Hospital, Sweden;
2
Department of Pediatrics, Ryhov County Hospital, Sweden
Objectives: Specific histopathologic changes in the small intestine,
which normalize during a gluten-free diet, are characteristic for
celiac disease. The histopathologic assessment of these changes
in duodenal biopsies is usually routine, but can be problematic. In
this study we investigated gene expression profiling as a
diagnostic tool for celiac disease as well as for monitoring
remission upon gluten free diet. Methods: 109 genes were
selected to reflect features that are characteristic of celiac
disease, i.e. alterations in crypt-villi architecture, inflammatory
response, and intestinal permeability. Selected genes were
examined for differential expression in normal mucosa compared
to celiac disease mucosa (histologically graded according to the
modified Marsh scale) in pediatric patients investigated for celiac
disease. The ability to classify biopsies using gene expression
was addressed by means of discriminant analysis. Results: Fifty
genes were differentially expressed, of which eight genes
(APOC3, CYP3A4, OCLN, MAD2L1, MKI67, CXCL11, IL17A, and
CTLA4) discriminated normal mucosa from celiac disease mucosa
without classification errors using leave-one-out cross-validation
(n = 39), and identified the degree of mucosal damage, divided
into groups Marsh 0, Marsh 2 - 3B and Marsh 3C. Validation using
an independent set of biopsies (n = 27) resulted in four discrepant
cases. Biopsies from two of these cases showed a patchy
distribution of lesions, indicating that discriminant analysis based
on single biopsies failed to identify celiac disease mucosa. In the
other two cases, serology (levels of anti-gliadin and anti-tissue
transglutaminase antibodies) supports class according to
discriminant analysis and histologic specimens were judged
suboptimal but assessable. The posterior probabilities of a single
Marsh 1 specimen were strikingly distributed between the Marsh 0
class and the Marsh 2 - 3B class, thus suggesting the possibility of
distinct classification. Conclusion: Gene expression profiling
shows promise as a diagnostic tool and for follow-up of celiac
disease. A classification based on gene expression analysis of
three biopsies per patient would entail a laboratory service cost
comparable to that of a histopathologic assessment. The
classification functions, however, need to be established using a
larger cohort of patients, and further validated.
P075
Allele specific gene expression from single cell RNAseq
Thomas Rygus1, Catalin Barbacioru4, Fuchou Tang2,3, Ellen
Nordman4, Kaiqin Lao4, M. Azim Surani2
1
Life Technologies, Germany; 2Wellcome Trust/Cancer Research
UK Gurdon Institute of Cancer and Developmental Biology,
University of Cambridge, Tennis Court Road, Cambridge, CB2
1QN, UK; 3BIOPIC, School of Life Sciences, Peking University,
Beijing, 100871, China; 42Genetic Systems, Applied Biosystems,
part of Life Technologies, 850 Lincoln Centre Drive, Foster City,
CA 94404, USA
The stochastic gene expression has the potential to determine the
phenotype of a cell. However it has never been globally shown in
mammalian cells in vivo under physiological conditions. Using
single cell RNA-Seq analysis, here we proved that up to 2,000
genes in individual cells of the mouse early embryos show allelic
differential expression. This is compatible with the fact that
regulation feedback by the protein product on its own transcription
usually can not discriminate between the two alleles of a same
gene, which will not damper the differential expression of the two
alleles. For all of these genes, their allelic differential expression is
at least partially determined by genetic polymorphisms since one
of the two alleles of the same gene always express lower than the
other one in both blastomeres from the same two-cell embryo.
This indicates that the subtle genetic polymorphisms between the
two alleles of a same gene can definitely determine the functional
readout of the gene in cis through either directly regulate the
transcription machinery or indirectly regulate the epigenetic status
of them in a same cell. At the same time, 1.6% of genes (or up to
200 genes) show allelic specific stochastic expression, that is, the
expression ratio between the two alleles of the same gene are
clearly different in the two essentially identical blastomeres of the
same two-cell embryo. This proves that stochastic gene
expression exists in mammalian early embryos, which can affect
expression of up to hundreds of genes significantly, and the
combination of deterministic and stochastic expression of the
genes will potentially regulate the precise fate, developmental
potential, and phenotype of a cell.
HRM & Methylation Studies
P076
The EpiQ Chromatin Analysis Kit: A New Tool for
Epigenetic Research
Viresh Patel
Bio-Rad Laboratories, United States of America
Eukaryotic DNA can exist in two general states, euchromatin
where the DNA is loosely packaged, accessible and
transcriptionally competent and heterochromatin where the DNA is
tightly packaged, inaccessible and transcriptionally silent.
Epigenetics controls the transition between these two states.
There are two main epigenetic events, DNA methylation and
histone modification which help regulate the chromatin state and
most current epigenetic analyses study either DNA methylation or
histone modification events. We have developed the EpiQ assay,
a new and fundamentally different product that assesses
chromatin structure in a quantitative manner and interrogates the
functional consequence of epigenetic events.
P077
Chloroplast DNA heterogeneity detection with HRM
analysis
Irem Uzonur, Esma Ozsoy, Zeynep Katmer, Fatih Koyuncu,
Gamze Akdeniz, Ali Rıza Atasoy
Fatih University, Turkey
Chloroplast DNA (cpDNA) has highly conserved regions and also
intergenic spacers with relatively high levels of polymorphisms for
the analysis of intraspecific to intergeneric levels of variation.
Chloroplast DNA (cpDNA) is a source of original markers very
useful in phylogeny and in population genetics. The degree of
universality of cpDNA primers within the plant kingdom varies. In
our case primers (KLR-F: 5’-AGTTCGAGCCTGATTATCCC-3’and
KLR-R: 5’-GCATGCCGCCAGCGTTCATC-3’) amplify a 298 bp
highly conserved region that is virtually a conserved region from
any land plant and many alga with end-point detection. We further
our end-point detection using qPCR assisted with HRM analysis
and sequencing to detect the genetic heterogeneity of cpDNA that
is undetectable with end-point PCR and agarose gel
electrophoresis from terrestrial plants: Urtica dioica, Zea mays,
Phaselous vulgaris and bloom forming micro-algae species
Prorocentrum minimum and Skeletonema costatum. In this
context as approved by sequencing, HRM assisted qPCR is a very
practical and sensitive tool that can be adapted to the different
levels of genetic heterogeneity determinations.
P078
Semi-quantitative analysis of O6-methylguanine-DNA
methyltransferase (MGMT) promoter hypermethylation
by high resolution melting (HRM) real-time PCR
Kok-Siong Poon1, Lily Chiu1, Evelyn Siew-Chuan Koay1,2
1
Molecular Diagnosis Centre, National University Hospital,
Singapore; 2Department of Pathology, Yong Loo Lin School of
Medicine, National University of Singapore
Objectives: O6-methylguanine-DNA methyltransferase (MGMT)
promoter hypermethylation has been reported as a good
prognostic biomarker for predicting favorable outcome in patients
with glioblastomas on alkylating agent chemotherapy. A reliable
prognostic test is able to accurately determine the extent of
promoter site methylation which is associated with epigenetic
silencing of the gene. Most commonly used methylation-specific
PCR assays are based on methylation-specific primers binding to
and interrogating the methylation status of a limited number of
CpG sites within the promoter sites of the genes. A high resolution
melting (HRM) real-time PCR has been developed on the
LightScanner 32 system, to serve as a rapid and inexpensive
method to detect and semi-quantify the full methylation of targeted
CpG sites in the MGMT promoter region. Methodology: To create
methylated and unmethylated controls, Cp Genome Universal
Methylated DNA (CHEMICONTM, Millipore Corporation, USA) and
genomic DNA prepared from lymphocytes were bisulfite-converted
with EZ DNA Methylation-Gold KitTM (Zymo Research, CA, USA),
respectively. The methylated DNA (metDNA) was mixed with
unmethylated DNA (unmetDNA) in metDNA:unmetDNA ratios of
80%, 60%, 40%, 20% and 10%. Amplification was performed with
a set of methylation-independent primers in the LS Mastermix with
incorporated LC Green dye (Idaho Technology Inc., UT, USA),
and the PCR assay conditions were optimized. The extent of
methylation of the 108bp amplicon, which encompasses 12 CpG
sites in the MGMT promoter region, was determined using the
proprietary HRM LS32 Amplicon Genotyping software. Seven
human cancer cell lines, namely MCF10A, BT549, SKBR3,
SW480, BT474, WM3899 and MCF12A were evaluated for their
MGMT methylation status using this assay. By using the melt
profiles of the above-mentioned methylation standards as
reference, the ratios of metDNA to unmetDNA were semiquantifiable within the intervals of <20%, 20%-40%, 40%-60%,
60%-80% and >80%. Results: The 100% fully methylated and
100% unmethylated DNAs controls
exhibited visually
distinguishable melt peaks at 80°C and 85°C, respectively. The
normalized melting curves showed a reproducible dose-response
relationship for different percentages of fully methylated DNA. Five
of the evaluated cell lines (MCF10A, BT549, SKBR3, BT474 and
MCF12A) did not show any methylation; the remaining two
(SW480& WM3899) showed positive results with 40-60% metDNA
and 100% metDNA, respectively. The assay can consistently
detect as low as 10% of metDNA in a mixture with 90%
unmetDNA. Conclusions: The HRM assay allows the rapid
screening and semi-quantification of full methylation of 12 targeted
CpG sites in the MGMT promoter region.
P079
Application of High Resolution Melting to SNP
genotyping of Porcine Ryanodine Receptor Gene
ANNA CASTELLO1, ANNA MERCADE2, ARMAND SANCHEZ1,2
1
Departament de Ciencia Animal i dels Aliments, Universitat
Autonoma de Barcelona, 08193 Bellaterra, Spain; 2Servei
Veterinari de Genetica Molecular, Facultat Veterinaria, Universitat
Autonoma de Barcelona, 08193 Bellaterra, Spain
High Resolution Melting Analysis (HRM) is a recently developed
technique for fast and cost-effective SNP genotyping based on the
analysis of the melting profile of PCR products, using dsDNA
intercalating dyes to monitor the reduction of fluorescence that
occurs from unmelted to melted DNA. A single nucleotide
polymorphism (SNP) in the porcine ryanodine receptor (ryr1) gene
was associated with malignant hyperthermia (MH) disease. This
disease causes important losses in swine industry due to
manifestation of MH post-mortem signals in susceptible animals
that affect detrimentally meat quality. As ryr1 mutation plays a key
role in animal production, it is of main importance to implement a
fast and economical method of diagnosis. An HRM protocol has
been developed to genotype the C/T substitution at nucleotide
1843 of ryr1 gene. With the EcoTM Real-Time PCR System and
using the high sensitive “release-on-demand” dye EvaGreen® we
have analysed DNA samples extracted from swine blood with
different methods in order to test the effects of genomic DNA
quality on melting behaviour. As it is well-known, inhibitors
carryover from the template could result in low sensitivity, poor
reproducibility and incorrect genotype. To validate results
obtained, all tested samples were previously genotyped by
pyrosequencing. The HRM technique presented in this study to
ryr1 genotyping is sensitive enough to allow the detection of the
C/T transition. Furthermore, DNA quality obtained with the
different extraction methods tested has not been a limiting fact.
Obtaining DNA samples as concentrate as possible after
purification, which will be diluted before HRM assay, overcomes
inhibitors carryover problems present in a low quality DNA. The
HRM assay for ryr1 described here offers clear advantages over
the PCR-RFLP methodology published by Fujii et al. (1991).
Moreover, HRM requires less optimization than similar systems
based on TaqMan and fluorescence resonance energy transfer
(FRET) probes. Compared to these methods HRM is a simpler
and more cost-effective way to characterize multiple samples.
Identification of human ryr1 SNPs using HRM has been also
described by Grievink and Stowell (2007). In conclusion, the use
of the HRM assay for porcine ryr1 genotyping considerably
reduces hands-on-time and costs. In addition, it is a closed assay
system requiring no post-PCR processing and results are
comparable to conventional methods.
P080
DNA methylation biomarkers for noninvasive detection
of prostate cancer
Kristina Daniunaite1, Feliksas Jankevicius2,3, Juozas Lazutka1,
Sonata Jarmalaite1
1
Faculty of Nature Sci., Vilnius University, Lithuania; 2Faculty of
Medicine, Vilnius University, Lithuania; 3Vilnius University Hospital
Santariskiu Clinics, Vilnius, Lithuania
Introduction. Prostate cancer (PCa) is the second most prevalent
malignancy of males characterized by high mortality rates. PCa
can be effectively treated if it is diagnosed in its early stages,
when the tumor is still confined to the prostate. Recently, aberrant
DNA methylation in promoters of tumor suppressor genes has
been proposed as one of the most important events in prostate
carcinogenesis. It occurs at early stages of tumor development
and can be detected by noninvasive means, including analysis of
urine sediments from PCa patients. Methods. For the development
of noninvasive molecular biomarkers of PCa aberrant promoter
methylation was analyzed in DNA from 102 samples of urine
sediment from previously untreated cases of biopsy-proven early
or medium stage PCa and 5 cases of benign prostatic hyperplasia
(BPH). Quantitative methylation-specific PCR (QMSP) was used
for sensitive detection of methylated DNA in catheterized urine
specimens. Genes GSTP1 (glutathione S-transferase pi 1),
RASSF1 (Ras association (RalGDS/AF-6) domain family member
1), and RARB (retinoic acid receptor beta), proven to be frequently
hypermethylated in prostate cancer, were chosen for this analysis.
The level of promoter methylation for particular gene was
evaluated by calculating percentage of methylated reference
(PMR) using ACTB as endogenous control. Results. At least one
of the three genes was hypermethylated in urine sediments in 97
of 102 PCa cases (95%), and 33 of 102 (32%) samples were
positive for methylation of at least two genes. RASSF1 was
methylated in 94% (93 of 99), RARB – in 31% (32 of 102), and
GSTP1 – in 14% (14 of 97) of the specimens. The average PMR
for positive cases was 40%, 5%, and 5% for RASSF1, RARB, and
GSTP1, respectively. High level of methylation (PMR >50%) was
detected in 35 of 93 (38%) cases for RASSF1, while PMR value
for RARB and GSTP1 reached only 31% and 17%, respectively.
Hypermethylation at relatively lower levels was also detected in
urine sediments from BPH patients – average PMR was 20%,
<1%, <1% for RASSF1, RARB, and GSTP1, respectively.
Conclusions. Preliminary results of our study show high sensitivity
of particular DNA methylation biomarkers for early and
noninvasive detection of prostate cancer.
P081
Does DNA methylation of the Estrogen Receptor Alpha
gene contribute to transcriptional regulation in the
bovine endometrium?
Rainer W. Fürst, Heinrich H.D. Meyer, Susanne E. Ulbrich
Germany
Among the reproductive tissues, the endometrium undergoes
functional changes during the estrous cycle which are regulated
by oestradiol-17β (E2) via the estrogen receptor alpha (ESR1).
We asked whether differential methylation of an intragenic region
of this receptor plays an important role in its transcriptional
regulation during both estrous cycle and early pregnancy in
bovine. Therefore, we monitored the mRNA expression of ESR1
and investigated associated global and local DNA methylation
patterns from heifers slaughtered at estrus or 18 days after estrus
(day 18 control), or during early pregnancy (day 18 - pregnant)
(n=6 each). In addition, white blood cells from cyclic heifers were
analyzed (n=6). Global DNA methylation was analyzed by the
luminometric methylation assay (LUMA). Using high resolution
melt (HRM) analysis, a specific area of the intron 1 region
composed of 16 CpG sites was amplified and subsequently
analyzed by pyrosequencing granting single CpG-site resolution.
The endometrium was only weakly methylated (6 % average
methylation). Neither HRM nor pyrosequencing of individual CpG
sites revealed a significant difference between the analyzed
groups of endometrial samples. In contrast, leucocytes with only
minute amounts of ESR1 transcripts showed a higher degree of
global methylation (83 % versus 76 % average methylation in
endometrium, respectively) and also a significantly higher
methylation over the analyzed CpG sites (31% average
methylation). In conclusion, DNA methylation of the analyzed
region of endometrial ESR1 seems not to be underlying the
transcriptional regulation during early pregnancy or cycle, although
it might contribute to appropriate tissue-specific expression.
P082
Microarray gene expression and mtDNA deletion
analysis in laser dissected human muscle fibers.
Matthias Elstner1, Konrad Olszewski1, Christoph Laub1, Anne
Averdam1, Lena Bruns1, Andreas Bender1, Holger Prokisch2,
Thomas Klopstock1
1
Ludwig-Maximilians University Munich, Germany; 2Helmholtz
Zentrum Munich, Germany
Background: Mitochondrial DNA (mtDNA) deletions cause
progressive external ophthalmoplegia (PEO), Kearns-Sayre
syndrome (KSS), and Pearson syndrome. Single size deletions
(SD) mostly occur sporadically in the germline, whereas mutations
of the nuclear-encoded mitochondrial replication machinery show
mendelian transmission and cause multiple deletions (MD) of
various lengths. The phenotypic spectrum of mtDNA deletion
syndromes is highly variable, ranging from mild myopathy to the
fatal multisystem KSS. This heterogeneity is considered to depend
on the inter- and intraindividual variance of tissue deletion load, as
well as nuclear disease modifiers and environmental factors.
Methods: We analyzed gene expression patterns of isolated
muscle fibers from nine patients with PEO (5 female / 4 male, 6
SD / 3 MD) using laser capture microdissection (LCM). By
exploiting the characteristic mosaic pattern of muscle tissue with
cytochrome oxidase (COX)-positive and -negative fibers, we
analyzed deletion load and gene expression patterns in fibers with
and without relevant OXPHOS dysfunction. Following RNA
isolation and in vitro transcription, we determined whole genome
expression patterns (Illumina®, WG6-V3) and examined
expression changes using pathway analysis (Ingenuity®). Results:
Deletion load reached a mean of 34% in COX-positive cells and
60% in COX negative fibers (p=0.002). Microarray analysis
identified 138 significantly regulated genes (Benjamini & Hochberg
false discovery rate 1%). Network analysis classified molecules as
involved in regulation of DNA replication, recombination and
repair. Furthermore, top canonical pathways include folic acid
synthesis, protein ubiquitination and autophagy. Discussion: By
using frozen human muscle biopsies and LCM, this methodology
may best approximate ‘in vivo’ transcriptome changes in
mitochondrial deletion syndromes. By sampling fibers with normal
and disrupted OXPHOS function from the same individual,
confounding variables, such as age, sex, deletion type,
heterogeneity and disease progression are abolished. We discuss
the significance of our findings in the light of published data and its
relevance regarding the understanding of disease mechanisms
and therapy. Since deletions occur age-dependent in muscle and
neurons, this data might also be of relevance for the
understanding of sarcopenia and neurodegenerative disorders.
P083
From traditional RAPD-PCR to next generation HRM
assisted real-time RAPD PCR
Irem Uzonur, Zeynep Katmer, Esma Ozsoy, Fatih Koyuncu,
Tugba Senel
Fatih University, Turkey
Traditional random amplified polymorphic DNA (RAPD) technique
is based on the amplification of DNA fragments using a short
arbitrary primer that anneals multiple locations on the genomic
DNA. This is followed by separation of amplified fragments based
on their sizes using gel electrophoresis. Samples are identified by
comparing the DNA bands of the end-point fingerprints, which are
expected to be consistent for the same primer, DNA and
experimental conditions used. The real-time PCR system is based
on the detection and quantitation of a fluorescent reporter. This
signal increases in direct proportion to the amount of PCR product
in a reaction. By recording the amount of fluorescence emission at
each cycle, it is possible to monitor the PCR reaction during
exponential phase where the first significant increase in the
amount of PCR product correlates to the initial amount of target
template. In addition, fluorescence data can be collected directly
from a real-time PCR instrument avoiding the drawbacks of end-
point detection. DNA melting analysis uses a double-stranded
DNA fluorescent dye to detect single nucleotide polymorphisms
(SNPs) and to perform mutation scanning following the
polymerase chain reaction (PCR) for DNA amplification. High
Resolution Melt or HRM analysis is a powerful technique for the
detection of mutations, polymorphisms and epigenetic
comparisons through differences in melting plots of amplicons by
measuring the melting temperature of amplicons in real time,
using a fluorescent DNA-binding dye. In this study, a novel RAPDPCR based method will be discussed to detect various levels of
genetic variation exploiting the advantages of various nextgeneration qPCR applications such as new dye Technologies and
HRM analysis.
P084
High Resolution Melting with Unlabelled Probe
approach for the analysis of Short Sequence Tandem
Repeats loci to sub-type Mycobacterium avium subsp.
paratuberculosis
Matteo Ricchi, Gianluca Barbieri, Norma Arrigoni
IZSLER, National Centre for Paratuberculosis, Piacenza, Italy
Mycobacterium avium subsp. paratuberculosis (Map) is the
causative agent of paratuberculosis in ruminants and other
species. Many methods have been proposed to sub-type Map:
multiplex PCR for IS900 integration loci, IS900 restriction fragment
length polymorphism, amplified fragment length polymorphism,
Pulsed Field Gel Electrophoresis and genotyping microarrays (1,
2). However, at the moment, methods based on the amplification
of Micro- and Mini- satellites tandem-repeats loci are considered
the emerging techniques for Map typing (1). In this regard, Short
Sequence Tandem Repeats loci (SSR1, SSR2, SSR8 and SSR9)
showed the highest discriminatory power; however, to identify
these loci, the direct sequencing of the amplicons is required. Our
aim was developing alternative methods to the sequencing. We
focused on SSR8, which is constituted by triplets ranging from
three to six repetitions. To identify each allele, we developed a
method based on an asymmetric qPCR, followed by HighResolution Melting (HRM) analysis with unlabelled probes. In
order to improve the efficiency of asymmetric PCR reaction, we
designed primers accordingly to the Linear-After-The-Exponential
(LATE) PCR strategy, while to avoid any elongation during the
amplification, the unlabelled probe was blocked adding C6-amino
group at its 3’ end. All PCR reactions were carried out on StepOne
Plus system (Applied Biosystems, Milan, Italy) in a final volume of
25 ml using LCGreen® Plus+ Melting Dye (Idaho technology Inc,
Salt Lake City, USA) and Right-Taq (EuroClone, Pero, Italy). HMR
analysis was carried out according to Palais (3), without ROX
normalisation and smoothing the graphs with running average
method. For the development of the technique we used three Map
strains containing, respectively, three, four and five repetitions; for
the allele with six repetitions, due to its rarity, we synthesised an
artificial amplicon. HMR analysis showed two melting domains for
each sample: one relative to the amplicon omoduplex (DNA
double strand) and another to the heteroduplex DNA single strand
/probe. The omoduplex domain did not allow any differentiation
between the various alleles, while the heteroduplex domain
showed almost 3 °C between each allele, permitting an unbiased
identification. These data were confirmed analysing 40 Map field
strains. The method herein proposed is robust, reproducible, cost
effective and faster than direct sequencing and could be usefully
applied to resolve locus SSR8.
1.Motiwala AS, Li L, Kapur V, Sreevatsan S., Current understanding of the
genetic diversity of Mycobacterium avium subsp. paratuberculosis. 2006.
Microbes Infect. 8, 1406-18.
2.Pribylova R, Kralik P, Pavlik I., 2009. Oligonucleotide microarray
technology and its application to Mycobacterium avium subsp.
paratuberculosis research: a review. Mol Biotechnol. 42, 30-40.
3.Palais R and Wittwer CT. 2009. Mathematical algorithms for highresolution DNA melting analysis. Methods Enzymol.454:323-43.
P087
RNAi: microRNA – siRNA Applications
P085
Titer dependent cytotoxicity of viral vectors measured
in real time via ECIS-technology
Jakob Müller1, Christian Thirion2, Pfaffl Michael W.1
1
Technische Universität München, Germany; 2Friedrich-BaurInstitute, Klinikum der LMU, Department of Neurology, Germany
Recombinant viral vectors are widespread tools for transfer of
genetic material in various modern biotechnological applications
like functional studies, vaccine development, gene therapy or RNA
interference. However the practical handling often bears crucial
problems. An accurate and reproducible titer assignment
represents the basic step for most downstream applications of
viral vectors not only considering precise MOI adjustment. As
necessary scaffold for the studies described in this work we
developed a qPCR based approach for viral particle
measurement. Proximate a fundamental vector originated problem
concerning physiological effects is that the appliance of viral
vectors can be attended by toxic effects on the individual cell
culture model or tissue. To determine the individually critical viral
dose we utilize Electric Cell-substrate Impedance Sensing (ECIS)
to reveal toxic effects leading to cell death. It was first introduced
in cell culture by Giaever and Keese and can be utilized for
examination of cell growth, cell motility, cell barrier function, in
vitro toxicology and is even applied in cancer research. With ECIS
technology the impedance change of a current flow through the
cell culture medium in an array plate is measured in a noninvasive manner. The device visualizes effects on cellular level
like cell attachment, cell-cell contacts or proliferation. Here we
describe the potential of this online measurement technique in an
in vitro model using the porcine ileal ephithelial cell line IPI-2I in
combination with an adenoviral transfection vector (Ad5 derivate).
With this approach we can show a clear dose-depending toxic
effect. The amount of applied virus correlates with the level of cell
death. So this assay offers the possibility to discriminate the
minimal non toxic dose of the individual transfection method.
Silencing of a Superoxide Dismutase in Phaedon
cochleariae (Coleoptera: Chrysomelidae) by using
RNAi.
René Roberto Bodemann, Wilhelm Boland, Antje Burse
Max Planck Institute for Chemical Ecology, Germany
RNA interference (RNAi) is a well conserved mechanism of
translation-inhibition in virtually all eucaryotic organisms. It gives
the opportunity to down regulate a single protein by using its
nucleotide sequence for synthesis of the interference-triggering
double stranded RNA (dsRNA) and observe effects of the
absence in vivo. Our model organism for checking the ways RNAi
could probably work in chrysomelid beetles, is P. cochleariae. As
a member of the subfamily Chrysomelinae he exhibits nine pairs
of dorsal accessory glands from which it can excrete droplets of
defense secretion in cases of disturbance. The deterrents are
either sequestrated and modified plant glucosides, de novo
produced iridoids or derive from a mixture of both. The transport
proteins and enzymes which make these three systems work are
the future targets of our RNAi. I started by silencing the
superoxide dismutase (SOD) that has very high expression level
in the glands which makes it easy to observe. The enzyme,
transported into the defense secretion, catalyses the reaction of
oxygen radicals, which are byproducts of deterrent-synthesis, to
hydrogen peroxide. Different amounts of dsRNA corresponding to
SOD sequence, have been injected in different life stages of
P.cochleariae and the expression-level of this SOD was analyzed
by qPCR. Up to now I found out, that injections into middle instars
larvae lead to a 90 to 99% reduction of SOD mRNA level
compared to control-groups which had been injected with the
same amount of not effecting GFP-dsRNA. Injections into pupae
or adults lead to similar effects. Interistingly following generations
was not affected in comparable dimensions. The next steps will be
to analyze the correlation of qPCR results with protein
concentrations and the search for an increased RNAi-effect in
offspring’s of pupae and adults. Furthermore siRNA experiments
will be done and an alternative delivery method for the dsRNA will
be investigated.
P088
P086
Specific and sensitive quantitative RT-PCR of miRNAs
with DNA primers.
Susanna Cirera1, Ingrid Balcells2, Peter K. Busk3
1
Genetics and Bioinformatics, University of Copenhagen, LIFE,
Denmark; 2Unitat de Ciència Animal, University Autonoma of
Barcelona, Spain; 3Section for Sustainable Biotech, Aalborg
University, Denmark
MicroRNAs are important regulators of gene expression and it is
of great interest to quantitatively determine their expression level
in different biological settings. Here, we describe a PCR method
for quantification of microRNAs based on a single reverse
transcription reaction for all microRNAs combined with real-time
PCR with two, microRNA-specific DNA primers. Primer annealing
temperatures were optimized by adding a DNA tail to the primers
and could be designed with a success rate of 94 %. The method
was able to quantify synthetic templates over eight orders of
magnitude and readily discriminated between microRNAs with
single nucleotide differences. Importantly, PCR with DNA primers
yielded significantly higher amplification efficiencies of biological
samples than a similar method based on locked nucleic acidsspiked primers, which is in agreement with the observation that
locked nucleic acid interferes with efficient amplification of short
templates. The higher amplification efficiency of DNA primers
translates into higher sensitivity and precision in microRNA
quantification. In conclusion, miR-specific quantitative RT-PCR
with DNA primers is a highly specific, sensitive and accurate
method for microRNA quantification.
Broad based profiling ~1150 microRNA designed in
version 16 of miRBase in a single experiment using
the SmartChip System from WaferGen Biosystems
Tony Dodge, alan chang, Harita Veereshlingam, Sangeetha
Anandakrishnan, Sherry Wei, Roy Bohenzky
WaferGen Biosystems, United States of America
The SmartChip Real-Time System from WaferGen Biosystems is
a platform for performing massively parallel real-time PCR assays
for multiple applications, including gene expression studies. This
system combines the benefits of real-time PCR, including
sensitivity, precision and dynamic range, with the low cost per
sample and high throughput profiling power of hybridization
arrays. The system consists of three components; a SmartChip,
comprising 5184 nanowells preprogrammed with gene-specific
reaction content, a SmartChip Nanodispenser for applying sample
and reaction mix to the SmartChips, and a SmartChip Cycler for
performing and collecting data from the real-time PCR assays.
Using cDNA as an input for this system, one can generate
thousands of data points which can comprise a thorough portrait
of relative gene expression in a sample. The mirnome has been
gaining increasing focus as a target population of RNA in
biomarker profiling. The discovery tools available for profiling
include labor intensive or limited accuracy methods in next
generation sequencing and hybridization arrays. Quantitative PCR
has been used extensively for profiling 100’s of microRNA on a
few samples, but these methods can both be costly and provide
only singlicate data points, arguable only a qualitative tool for
microRNA profiling. Here we present a cost effective, broadly
defined and quantitative microRNA profiling tool in the SmartChip
Human microRNA Panel (version 2). A new ligation based method
is combined in a SYBR® Green quantitative PCR method. Over
1150 microRNA assays have been designed to miRBase v16,
allowing for a cost effective quantitative profiling tool with each
assay in quadruplicate per panel. We will describe the new
chemistry, its performance with respect to sensitivity and
specificity, and representative data in control tissues.
P089
ChIP-qPCR and qbasePLUS jointly identify a MYCNactivated miRNA cluster in cancer
Barbara D'haene1, Pieter Mestdagh2, Daniel Muth3, Frank
Westerman3, Frank Speleman2, Jo Vandesompele1,2
1
Biogazelle, Zwijnaarden, Belgium; 2Center for Medical Genetics,
Ghent University, Ghent, Belgium; 3Department of Tumour
Genetics, German Cancer Center, Heidelberg, Germany
Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) is
very well suited to assess and quantify direct binding of specific
regulatory proteins to genomic DNA sequences. Unfortunately,
data-normalization and accurate quantification appear to be a
major challenge for many users. Here, we demonstrate that ChIPqPCR in combination with state-of-the-art real-time PCR dataanalysis software enables convenient and reliable quantification.
We applied ChIP-qPCR to assess binding of transcription factor
MYCN to miRNA cluster 17-92, to a positive control target, MDM2,
and to a negative control target region. ChIP-qPCR was
performed in two MYCN-overexpressing neuroblastoma cell lines
(IMR5 and WAC2) using SYBR Green I detection chemistry in a
384-well plate and signals were normalized based on the average
abundance of three non-specific genomic regions in the ChIP
samples using the qbasePLUS multiple reference gene
normalization technology. Fold enrichment was calculated relative
to the input sample (non-precipitated) and compared to that of a
fourth non-specific region (negative control target). Using this
approach we were able to demonstrate strong MYCN-binding to
the positive control and the miR-17-92 cluster. In keeping with this,
the expression level of the miR-17-92 cluster is substantially
increased in primary neuroblastoma tumor samples, in which the
MYCN gene is amplified and overexpressed.
The results confirm the power of ChIP-qPCR in combination with
the data-analysis software qbasePLUS to study gene regulation.
P090
LNA-based miRNA qPCR facilitates the discrimination
of different stages in cancer progression in a
transgenic mouse model
Kim B Barken1, Ditte Andreasen1, Rolf Søkilde1, Jan
Stenvang2, Niels Tolstrup1, Thomas Litman3, Boye S Nielsen1,
Peter Mouritzen1
1
Exiqon, Denmark; 2Copenhagen University, LIFE, Denmark;
3
Litman Consulting, Denmark
miRNAs are small RNAs (21-23nt) which function as posttranscriptional regulators of gene expression, possibly responsible
for regulation of more than one third of the cellular transcriptome.
Numerous studies provide evidence for a causal link between
miRNA dysregulation and several disease states, including human
cancers. We have developed the miRCURY LNA™ Universal RT
miRNA qPCR platform to facilitate sensitive and accurate miRNA
expression profiling in both human, mouse and rat. Here we have
used the new Pick&Mix product where a custom selection of
assays is supplied in ready to use PCR plates. We have applied
the miRCURY LNA™ Universal RT miRNA PCR platform to study
progression of breast cancer in a genetically modified mouse
model and show that the expression pattern of miRNAs clearly
provides a means to stage the cancer progression. In the study, a
group of wild type mice was compared with a group of polyoma
middle T antigen (PymT) transgenic mice. This mouse model
mimics the progression of human breast cancer and can be used
as an experimental model to investigate the mechanisms involved
in the process of metastasis. Samples were taken at different
progression stages of the tumor and a selection of 18 highly and
differentially expressed miRNAs were identified by miRNA profiling
on miRCURY LNA™ arrays. The expression of the 18 miRNAs
was investigated by miRCURY LNA™ Universal RT microRNA
PCR during three different progression stages of the cancer:
Adenomas (early), early carcinomas (intermediate) and
adenocarcinomas (late). Principal component analysis of the
expression of the 18 miRNAs clearly shows clustering of the early
tumor stage together with the wild type whereas both the
intermediate and the late stage clearly clusters separate from the
wild type. The results further confirm that the LNA-based miRNA
qPCR serve as an ideal platform for disease biomarker
identification.
Late submissions:
P091
Aurnhammer, Christine; Haase, Maren; Muether, Nadine;
Häusl, Martin; Huber, Ingrid; Nitschko, Hans; Busch, Ulrich;
Sing, Andreas; Erhardt, Anja; Baiker, Armin
Organization(s): Bavarian Health and Food Safety Authority
(LGL), Germany
Background: Viral vectors based on adeno-associated virus
serotype 2 (AAV2) constitute promising tools in human gene
therapy. The inverted terminal repeats (ITRs) within the viral
genome are the only cis-acting viral elements required for
functional AAV2 vector generation and constitute the lowest
common denominator of all AAV2-based vectors. However, so far,
no PCR-based method for the detection and quantification of
AAV2-ITRs could be established due to their extensive secondary
hairpin structure. Current PCR-based methods are therefore
predominantly targeting vector-encoded transgenes or regulatory
elements.
Methods: We established an AAV2-ITR sequence-specific
quantitative PCR (qPCR) method. Primers and BBQ-labeled probe
are located within a particular subregion of the ITR sequence and
have been designed to detect wild type AAV2 and AAV2-based
vectors.
Results: This method is suitable for the evidence of both, singlestranded (genomic) DNA derived from AAV2 vector particles and
double-stranded DNA derived from producer plasmids. The linear
dynamic range could be shown for 102 to 107 copies and the
detection limit determined as 50 copies. A practical approach for
the analysis of putative cross reactivities against closely related
AAV serotypes utilizing synthetic oligo nucleotides revealed some
cross reactivity against orthologous sequences of AAV1, 3, 6 and
7 but not against AAV4, 5, 8 and 9. For AAV vector production
adenovirus type 5 (Ad5) is often used in terms of its helper virus
properties. Therefore, we further investigated the specificity of our
qPCR method using Ad5-DNA and could prove the method to
result in no detectable cross reactivity with Ad5.
Conclusion: This method comprises the first qPCR system
facilitating the detection and quantification of AAV2 -ITR
sequences. Since this method can be applied for all AAV2-based
vectors in a “one for all”-based manner, it will significantly simplify
AAV2 vector genome titrations in the future.
List of participants
Afif Abdel Nour
LaSalle Beauvais
19, rue Pierre Waguet
60000 Beauvais France
[email protected]
Elisabeth Aberl
TUM Z I E L
85354 Freising Germany
[email protected]
Ivana Adams
QIAGEN
Qiagen Str. 1
40721 Hilden Germany
[email protected]
Amalia Adler-Beutgen
Fujifilm Europe GmbH
Heesenstr. 31
40549 Düsseldorf Germany
[email protected]
Raza Ahmed
LSCA, Lakeside, Cheadle Royal
Business Park
SK8 3GR Stockport United Kingdom
[email protected]
Peter Aigner
Invetech
Industriestraße 25
8604 Volketswil Switzerland
[email protected]
Javier Alba
Bio-Rad Laboratories
Garrotxa 10-12, 1 A1
08820 El Prat Spain
[email protected]
Andreas Albers
Roche Diagnostics GmbH - DAMP 6164
DAMP 6164
82377 Penzberg Germany
[email protected]
Ahmed Alharbi
Taibah University
P.O. Box 8766
21492 Jeddah Saudi Arabia
[email protected]
Christian Altenhofer
TUM
Urzenweg 26
4121 Altenfelden Austria
[email protected]
Ditte Andreasen
Exiqon
Skelstedet 16
2950 Vedbaek Denmark
[email protected]
Elisabeth Lill Andreassen
ArcticZymes AS
Sykehusveien 23
9294 Tromsø Norway
[email protected]
Birgit Anwald
Beckman Coulter Genomics GmbH
Am Neuland 1
82347 Bernried Germany
[email protected]
Hans Attig
QIAGEN
Qiagenstraße 1
[email protected]
Hubert Becher
Amplifa
Rickenbacher Strasse 107
88131 Lindau Germany
[email protected]
Herbert Auer
IRB Barcelona
Baldiri Reixac 10
08028 Barcelona Spain
[email protected]
Ingrid Bechler
Roche Diagnostics GmbH
Roche Diagnostics GmbH DAMP 6164
[email protected]
Madhu Augustine
Life Technologies
1149 Chess Drive
94404 Foster City United States
[email protected]
Christiane Becker
TUM
Liesel-Beckmann-Strasse 6
[email protected]
Christine Aurnhammer
Bavarian Health and Food Safety
Authority (LGL)
Veterinärstr. 2
85764 Oberschleißheim Germany
[email protected]
Beatriz Bellosillo
Hospital del Mar
Passeig Marítim 25-29
[email protected]
Jean-Christophe Avarre
Institut de Recherche pour le
Développement
IRD-GAMET, BP 5095
34196 Montpellier cedex 05 France
[email protected]
Ali Ayadi
Habib Bourguiba Hospital: Sfax - Tunisia
Laboratory of parasitology and
mycology:Habib Bourguiba Hospital
3029 Sfax Tunisia
[email protected]
Beatrix Bahle
Nonnewald 2
[email protected]
Stephan Bähler
QIAGEN Instruments AG
Garstligweg 8
CH-8634 Hombrechtikon Switzerland
[email protected]
Kim B Barken
Exiqon
Bygstubben 9
[email protected]
Ross Thomas Barnard
The University of Queensland
building 68, room 319
4072 Brisbane Australia
[email protected]
Christine Baumhauer
Daiichi Sankyo Europe GmbH
Lochhamerstr. 29 RG
82152 Martinsried Germany
[email protected]
Ingrid Bayer
Histologie Labor Bayer
Belgradstr. 8
80796 München Germany
[email protected]
Marc P. Beal
81 Digital Drive
94949 Novato United States
[email protected]
Stefan Bentink
Exosome Diagnostics GmbH
Am Klopferspitz 19a
[email protected]
Bajram Berisha
TUM Weihenstephan
Hittostr. 4
[email protected]
Tim Berschneider
Nonnenwald 2
[email protected]
Nadia Bertazzon
CRA-Centro di Ricerca per la Viticoltura
Viale XXVIII aprile, 26
31015 Conegliano Italy
[email protected]
Holger Berthel
Bioline GmbH
Im Biotechnologie Park TGZ 2
14943 Luckenwalde Germany
[email protected]
Arnaud BEURDELEY
ROCHE DIAGNOSTICS FRANCE
Lab Network - Biologie Moléculaire
38240 MEYLAN France
[email protected]
Daniela Bischof Boesch
Microsynth AG
Schützenstrasse 15
9436 Balgach Switzerland
[email protected]
Francisco Bizouarn
Bio-Rad Laboratories Inc
2000 Alfred Nobel Drive
94547 Hercules United States
[email protected]
Lotte Bjerrum
Danish Technological Institute
Kongsvang allé 29
8000 Aarhus C Denmark
[email protected]
Silvia Bobenstetter
LMU
Liebigstr.38
[email protected]
Nicolas BRUNET
2, avenue du Vercors
38240 MEYLAN France
[email protected]
Mark Chilcott
Bio-Rad
[email protected]
Marie-Dominique Bock
Sanofi-Aventis
1, avenue Pierre Brossolette
91385 CHILLY-MAZARIN France
[email protected]
Florian Buckel
MAB Discovery GmbH
Forstenriederstr. 8-14
82061 Neuried Germany
[email protected]
Lily Chiu
National University Hospital
5 Lower Kent Ridge Road
119074 singapore Singapore
[email protected]
Antje Burse
Max Planck Institute for Chemical
Ecology
Hans-Knoell-Str.8
D-07745 Jena Germany
[email protected]
Lai-on Chu
DiagCor Bioscience Inc. Ltd.
28/F, Tower A, Billion Centre,
Kowloon Hong Kong
[email protected]
René R. Bodemann
Ecology
Hans-Knoell-Str.8
D-07745 Jena Germany
[email protected]
Harry Boeltz
Fluidigm Europe
Parnassustoren
1076 AZ Amsterdam Netherlands
[email protected]
Manja Böhme
Biotype Diagnostic GmbH
Moritzburger Weg 67
01109 Dresden Germany
[email protected]
Elena Borzova
UEA
1 Cross Lane
NR3 1BU Norwich United Kingdom
[email protected]
Barts and the London School of Medicine
Academic Surgery
E1 1BB London United Kingdom
[email protected]
Silvia Calatroni
A.O. Treviglio-Caravaggio
via Folla di Sotto 11
27100 Pavia Italy
[email protected]
Fabrizio Carletti
Inmi Lazzaro Spallanzani
Via Nicola Laurantoni 54
00149 Rome Italy
[email protected]
Hanna Bragde
Länssjukhuset Ryhov
Building E3 Level 4
55185 Jönköping Sweden
[email protected]
ANNA CASTELLÓ
UNIVERSITAT AUTONOMA DE
BARCELONA
FACULTAT DE VETERINARIA
08193 BELLATERRA Spain
[email protected]
Maksim Bratchikov
Biosta Ltd.
S.Zukausko 18-117
08234 Vilnius Lithuania
[email protected]
Paolo Catarsi
IRCCS Policlinico San Matteo
Foundation, Piazzale Golgi
27100 Pavia Italy
[email protected]
Sabine Brauer
IMGM Laboratories GmbH
Lochhamer Str. 29
[email protected]
Inge Celler
TU München Weihenstephan
[email protected]
Bertram Brenig
University of Goettingen
Burckhardtweg 2
37077 Göttingen Germany
[email protected]
Vladimir Celmare
Roche Diagnostics Romania
3-5 Piata Presei Libere, City Gate
Building, South Tower, Floors:4A,5,6, 1st
District Bucharest
020335 Bucharest Romania
[email protected]
Irena Brinkmann
Institute of Transfusion Medicine and
Immunology
Ludolf-Krehl Strasse 13-17
68167 Mannheim Germany
[email protected]
Urska Cepin
National Institute of Biology
Vecna pot 111
SI-1000 Ljubljana Slovenia
[email protected]
Kai-Uwe Brodersen-van Aalst
Interleuvenlaan 12A
B-3001 Leuven Belgium
[email protected]
Alexander M Chagovetz
University of Utah
2315 East Sheridan Rd
84108 Salt lake City United States
[email protected]
Melanie Broszat
Uniklinik Freiburg
Breisacherstr. 115 b
79106 Freiburg Germany
[email protected]
Caifu Chen
Life Technologies
850 Lincoln Centre Dr.
[email protected]
Lindsay Chung
BIOKÉ
Plesmanlaan 1d
2333BZ Leiden Netherlands
[email protected]
Susanna Cirera
University of Copenhagen, LIFE
Groennegaardsvej 3,
1870 Frederiksberg Denmark
[email protected]
Philippe CLAIR
UNIVERSITE MONTPELLIER 2
Campus Triolet
34095 MONTPELLIER Cedex 5 France
[email protected]
Sabine CLEOPHAX
BIOCORTECH
8 rue Croix de Jarry
75013 PARIS France
[email protected]
Aron Cohen
Azure PCR
59A Brent Street
NW4 2EA London United Kingdom
[email protected]
Michelle Collins
2000 Alfred Nobel Dr
[email protected]
Ron M. Cook, Ph.D.
81 Digital Drive
[email protected]
Muriel CRAYNEST
EUROGENTEC
rue du Bois Saint Jean 5
4102 Seraing Belgium
[email protected]
Valentina Dall'Olio
Cogentech
via Adamello, 16
20139 Milan Italy
[email protected]
Benedicte Danis
UCB Pharma S.A.
Chemin du Foriest
1420 Braine-l'Alleud Belgium
[email protected]
Kristina Daniunaite
Vilnius University
M. K. Ciurlionio 21/27
LT-03101 Vilnius Lithuania
[email protected]
Zuzana Dobsakova
Cytogenetic Laboratory Brno
Veveri 39
602 00 Brno Czech Republic
[email protected]
Maxime Eugene
EUROGENTEC
Rue du Bois Saint Jean 5
[email protected]
Katrin Danowski
TU München
General-von-Stein-Str. 8a
[email protected]
Stefanie Dommel
TU München
Mühlenweg 1111
[email protected]
Patrick Fach
French Agency for Food, Environmental
and Occupational Health (ANSES)
23, avenue du général de Gaulle
94706 Maisons-Alfort France
[email protected]
Philip Day
University of Manchester
Manchester Interdisciplinary Biocentre
M1 7DN Manchester United Kingdom
[email protected]
Maxime Lilian Dooms
LaSalle Beauvais
Stagiaire DISC - LaSalle Beauvais
[email protected]
Inky de Baere
Biocartis
Anwerpsesteenweg 237
2350 Vosselaar Belgium
[email protected]
Roya Doroudi
Life Technologies
Frankfurther Str 129b
64293 Darmstadt Germany
[email protected]
Sabine Delannoy
Agence Nationale de Sécurité Sanitaire
(ANSES)
23 avenue du général de Gaulle
94706 Maisons-Alfort France
[email protected]
Brigitte Dötterböck
TU München
[email protected]
Dirk Demuth
Sigma-Aldrich Company Ltd.
84 Burnage Lane
M19 2WL Manchester United Kingdom
[email protected]
Holger Densow
Biometra GmbH
Rudolf-Wissell-Straße 30
D-37079 Göttingen Germany
[email protected]
Stefaan Derveaux
WaferGen Biosystems
7400 Paseo Padre Parkway
94555 Fremont United States
[email protected]
François-Xavier Desforges
genewave
le Dorian b2
75011 Paris France
[email protected]
Jan Detmers
Chimera Biotec GmbH
Emil Figge Str. 10
44227 Dortmund Germany
[email protected]
Barbara D'haene
Biogazelle
Technologiepark 3
9052 Zwijnaarde Belgium
[email protected]
Jeroen Dijkstra
Radboud University Nijmegen Medical
Centre
PO Box 9101
6500 HB Nijmegen Netherlands
[email protected]
Thu Hien Do
Katholieke Universiteit Leuven
Kasteelpark Arenberg 13, bus 2455
3001 Leuven Belgium
[email protected]
Mike Dyson
WaferGen Biosystems
7400 Paseo Padre Parkway
9455 Fremont United States
[email protected]
Christoph Eckert
Metabion
Lena-Christ-Str. 44
82152 Planegg-Martinsried Germany
[email protected]
Franck Edouard
Bio-Rad
3 boulevard Poincare
92430 Marnes La Coquette France
[email protected]
Marlene Eggert
Institut für Humangenetik
Goethestraße 29
[email protected]
Morten Elde
ArcticZymes As
Sykehusveien 23
9294 Tromso Norway
[email protected]
Matthias Elstner
Klinikum Großhadern
Marchioninistr. 15
81377 Munich Germany
[email protected]
Fons Elstrodt
BIOKÉ
Plesmanlaan 1d
[email protected]
Markus Epe
Affymetrix
Fürstenrieder Straße 273
[email protected]
Aurore Falcoz
38240 MEYLAN France
[email protected]
Netta Fatal
Thermo Scientific
Keilaranta 16A
02150 Espoo Finland
[email protected]
Richard Fekete
Life Technologies
2130 Woodward St.
78740 Austin United States
[email protected]
Eva Felder
Sanitätsakademie der Bundeswehr
Neuherbergstr.11
[email protected]
Jovita Fernandez-Pinero
Centro de Investigación en Sanidad
Animal (CISA-INIA)
Ctra Algete a El Casar, s.n
28130 Valdeolmos, Madrid Spain
[email protected]
Anna Ferrer
Center for Genomic Regulation - CRG
Doctor Aiguader, 8
[email protected]
LUISA FILIPPIN
CRA-VIT Centre for Research in
Viticulture
viale XXVIII Aprile 26
31015 CONEGLIANO Italy
[email protected]
Barbara Fitzky
Life Technologies
Frankfurter Str, 129B
[email protected]
Krzysztof Flisikowski
Technical University of Munich, WZW
Liesel-Beckmannstr. 1
[email protected]
Christine Fochtmann
TU München
[email protected]
Choy Len Fong
Life Technologies
Blk 33, #05-03
739256 Singapore Singapore
[email protected]
FRANCESCA FONTANA
SILICON BIOSYSTEMS SPA
VIA DEI LAPIDARI 12
40129 BOLOGNA Italy
[email protected]
Kelly Giles
Technology Networks Limited
Bull Lane Industrial Estate
CO10 0FD SUDBURY United Kingdom
[email protected]
David Kennard
Azure PCR
59A Brent Street
[email protected]
Yadvinder Gill
MARS, WALTHAM CENTRE FOR PET
NUTRITION,
LE14 4RT LEICESTERSHIRE United
Kingdom
[email protected]
Amin Forootan
Multid Analyses AB
Odinsgatan 28
41103 Göteborg Sweden
[email protected]
Roderic Alexander Fueerst
IT-IS Life Science Ltd
Bldg 1000, Unit 1201 & 1202
None Cork Ireland
[email protected]
Andreas Funke
IDT Biologika GmbH
Am Pharmapark
06861 Dessau-Rosslau Germany
[email protected]
Rainer W. Fürst
TUM
[email protected]
Hendrik Fuss
Molecular Machines & Industries
Breslauer Strasse 2
85386 Eching Germany
[email protected]
Mirco Geisler
Sigma Aldrich
Eschenstr.5
82024 Taufkirchen Germany
[email protected]
Katharina Gellrich
Physiology Weihenstephan
Ampertal 13
85777 Fahrenzhausen Germany
[email protected]
Lars Gerdes
Bayerisches Landesamt für Gesundheit
und Lebensmittelsicherheit (LGL)
Veterinästr. 2
D-85764 Oberschleißheim Germany
[email protected]
Matthew Gibbs
Mars Petcare
Waltham Centre for Pet Nutrition
LE14 4RT Melton Mowbray United
Kingdom
[email protected]
Alfons Gierl
Dekanat der Fakultät WZW
Alte Akademie 8
85354 Freising-Weihenstephan Germany
[email protected]
Christian Giersdorff
PensionCapital GmbH
Kantstraße 2
[email protected]
Winfried Girg
Qiagen GmbH
Qiagenstr. 1
[email protected]
Florian Glaser
Evotec AG
Schnackenburgallee 114
22525 Hamburg Germany
[email protected]
Viktoria Glunk
BioEPS/TUM
Gaymannstr. 1
[email protected]
Irene Görzer
Medical University of Vienna
Kinderspitalgasse 15
1095 Vienna Austria
[email protected]
Brigitte Gramsamer
InstMikroBioBw
Neuherbergstraße 11
[email protected]
Ursula Grepl
Nonnenwald 2
[email protected]
Elena Grigorenko
Life Technologies, Inc
12 Gill Street Suite 4000
01801 Woburn United States
[email protected]
Diana Grochova
Cytogenetic Laboratory Brno
Veveri 39
602 00 Brno Czech Republic
[email protected]
Anna E Groebner
Technische Universität München
[email protected]
Josef Gross
TUM
[email protected]
Cornelia Große
University Halle-Wittenberg
Kurt-Mothes-Str. 3
06120 Halle Germany
[email protected]
Helga Gruber
Authority
Veterinärstr. 2
[email protected]
Patrick Guertler
Authority
Veterinärstr. 2
[email protected]
Michele Guescini
University of Urbino
Via I Maggetti, 26
61029 Urbino Italy
[email protected]
Marina GUILLET
38240 MEYLAN France
[email protected]
Sandra GUILLOUD
38240 MEYLAN France
[email protected]
Stephen Gunstream
1710 Commercial Park
52241 Coralville United States
[email protected]
Mandana Haack-Sørensen
Rigshospitalet
Juliane Mariesvej 20, 9302
2100 Copenhagen Denmark
[email protected]
Heli Haakana
Ratastie 2
01620 Vantaa Finland
[email protected]
Gerd Haberhausen
Nonnenwald 2
[email protected]
Knut Hamann
Lifetechnologies
Theodor-Strom-Str. 15
23863 Bargfeld-Stegen Germany
[email protected]
Matthias Hanczaruk
Bundeswehr Institute of Microbiology
Neuherbergstr. 11
[email protected]
Rita Härteis
Nonnenwald 2
[email protected]
Luise Hartmann
TU Muenchen
Ehrenpreisstrasse 9
86899 Landsberg Germany
[email protected]
Karl H. Hasenstein
Univ. Louisiana Lafayette
300 E. St. Mary Blvd
70504-2451 Lafayette United States
[email protected]
Akihiro Hirano
RIKEN (The Institute of Physical and
Chemical Research, Japan)
2-1 Hirosawa
351-0198 Wako-city Japan
[email protected]
Andrea Hecker
Medical Faculty Mannheim, Heidelberg
University, Ludolf-Krehl-Strasse 13-17
[email protected]
Hans-Joachim Hoeltke
D-82377 Penzberg Germany
[email protected]
Torben Helledie
Exiqon
Skelstedet 16
[email protected]
Hans Hoenicka
Institute of Forest Genetics
Sieker Landstr. 2
22927 Großhansdorf Germany
[email protected]
Jan Hellemans
Biogazelle
Technologiepark 3
9052 Ghent Belgium
[email protected]
Gerhard Hofer
QIAGEN
QIAGEN Str.1
[email protected]
Katharina Heller
ZIEL PhD-Kolleg "Epigenetik"
Gregor-Mendel-Str. 2
[email protected]
Michael Hoffmann
Roche Diagnostics
Nonnenwald 2
[email protected]
Kaisa Helminen
Thermo Scientific
Keilaranta 16A
02150 Espoo Finland
[email protected]
Ingrid Hoffmann
Nonnenwald 2
[email protected]
Jana Hemmerling
PhD Kolleg "Nutritional adaptation and
epigenetic mechanisms"
[email protected]
Janin Hofmann
Ipal GmbH
Bundesallee 171
10715 Berlin Germany
[email protected]
Anna Henger
Biolytix AG
Benkenstr. 254
4108 Witterswil Switzerland
[email protected]
Neli Hevir
Faculty of Medicine
Vrazov trg 2
1000 Ljubljana Slovenia
[email protected]
Mirela Hila
Pediatric Hospital Sibiu
9-9A Dimitrie pompeiu Street, building
2A, ground floor
[email protected]
Alison Hills
GSTS Pathology, Guys and St Thomas
NHS Foundation Trust
Cytogenetics Department
SE1 9RT London United Kingdom
[email protected]
Chris Hingley
Primerdesign Ltd
Millbrook Technology Campus
SO15 0DJ Southampton United Kingdom
[email protected]
Kassie Hirani
Bioline
Unit 16 The Edge Business Centre
NW2 6EW London United Kingdom
[email protected]
Béatrice HOFMANN
EUROGENTEC
Rue du Bois Saint Jean
[email protected]
Lars Christian Hofmann
Clontech / Takara Bio
Guardinistr. 95
[email protected]
Karsten Höhn
Analytik Jena AG / Biometra GmbH
Konrad-Zuse-Straße 1
07745 Jena Germany
[email protected]
Barbara Holzer
Medical University of Vienna
Waehringer Guertel 18-20, 5Q
1220 Vienna Austria
[email protected]
Nikos Hontzeas
Sigma Aldrich
2909 Laclede Ave.
63103 St. Louis United States
[email protected]
Michaela Hoock
Lautengartenstrasse 6
4052 Basel Switzerland
[email protected]
Martin Horat
Roche Diagnostics AG
Forreenstraße
6343 Rotkreuz Switzerland
[email protected]
Martin Horlitz
QIAGEN GmbH
QIAGEN Strasse 1
[email protected]
Ralf Horres
GenXPro GmbH
Altenhöferallee 3
60438 Frankfurt am Main Germany
[email protected]
Eric Houpt
University of Virginia
University of Virginia
22947 Charlottesville United States
[email protected]
Ilona Hromadnikova
Third Faculty of Medicine, Charles
University Prague
Ruska 87
10000 Prague 10 Czech Republic
[email protected]
Ingrid Huber
Bayerisches Landesamt für Gesundheit
und Lebensmittelsicherheit
Veterinärstr. 2
[email protected]
YOUG MIN HUH
YONSEI UNIVERSITY
250 seongsanno, seodaemun-gu
120-749 seoul South Korea
[email protected]
André Imhof
Molecular Machines & Industries
Flughofstrasse 37
8152 Glattbrugg Switzerland
[email protected]
Peter Jacobs
Lifetechnologies
Suikerkaai 1
1500 Halle Belgium
[email protected]
Aurélie JAMIN
EUROGENTEC
5 rue du Bois Saint Jean
[email protected]
Greta Johannesson
TATAA Biocenter
Odinsgatan 28
41103 Gothenburg Sweden
[email protected]
Martin Judex
Kurt-von-Unruh-Weg 6
84085 Langquaid Germany
[email protected]
Katrin Juling
Eurofins Medigenomix GmbH
Anzinger Str. 7a
85560 Ebersberg Germany
[email protected]
Dafni-Maria Kagkli
European Commission-Joint Research
Centre
via Enrico Fermi 2749
21027 Ispra Italy
[email protected]
Olev Kahre
Solis BioDyne
Riia 185a, 51014 Tartu, Estonia
51014 Tartu Estonia
[email protected]
Sebastian Kaiser
LMU München
Ludwigstr. 33
[email protected]
Andreas Kalchschmid
Genetic DI (Europe) AG
Am Mittleren Moos 48
86167 Augsburg Germany
[email protected]
Niels Kaldenhoven
Genmab BV
Yalelaan 60
3584 CM Utrecht Netherlands
[email protected]
Gustav Karlberg
Illumina
Chesterford Research Park
CB10 1XL Saffron Walden, Essex United
Kingdom
[email protected]
Joachim Richard Karlsen
Danish Medicines Agency
Axel Heides Gade 1
2300 Copenhagen S Denmark
[email protected]
Kersti Kask
Solis BioDyne OÜ
Riia 185 a
50415 Tartu Estonia
[email protected]
Ly Käsper
Solis Biodyne OÜ
Riia 185a, 51014 Tartu, Estonia
51014 Tartu Estonia
[email protected]
Zeynep Katmer
Fatih University
Buyukçekmece
34500 Istanbul Turkey
[email protected]
Christine Kaufmann
Weihenstephaner Steig 23
[email protected]
Ian Kavanagh
Blenheim Road
TW2 5NH Epsom United Kingdom
[email protected]
Linda Veronique Kazandjian
MAB Discovery GmbH
Forstenriederstr. 8-14
[email protected]
Andreas Keck
Affymetrix
[email protected]
Minna Riikka Koivula
Thermo Fisher Scientific/Finnzymes Oy
Ratastie 2
[email protected]
Antje Kern
Affymetrix
[email protected]
Varaprasad Kolla
University Hospital, Basel
Hebelstr20
[email protected]
David Michael Kerry
Life Technologies
5791 Van Allen Way
92008 Carlsbad United States
[email protected]
Michal Konecny
St. Elizabeth Cancer Institute
81250 Bratislava Slovakia
[email protected]
HYE SEON KIM
YONSEI UNIVERSITY
250 seongsanno, seodaemun-gu
120-749 seoul South Korea
[email protected]
Regina Konrad
Authority
Veterinärstr. 2
[email protected]
Seong-Youl Kim
Bioneer Corporation
49-3 Munpyung-dong, Daeduck-ku
306-220 Daejeon South Korea
[email protected]
Mirjana Kozulic
QIAGEN Instruments AG
Garstligweg 8
8634 Hombrechtikon Switzerland
[email protected]
Benedikt Kirchner
Giggenhauser Straße 1
[email protected]
Vanessa Kraft
Lange Point 13/101
[email protected]
Christoph Kirsch
Macherey-Nagel GmbH & Co.KG
Neumann-Neander-Straße 6
52355 Düren Germany
[email protected]
Hannes Kirzinger
Roche
Fritz-Schlamppstr. 5
86932 Pürgen Germany
[email protected]
Jörg Kleiber
Hochschule Weihenstephan
Hochschule Weihenstephan
[email protected]
Manfred Kliem
Roche Diganostics Deutschland GmbH
Sandhofer Str. 116
[email protected]
Heike Kliem
TUM, Weihenstephaner Berg 3
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Thilo Kluetsch
Agilent
Sindesldorfer Strasse 60e
[email protected]
Kevin L Knudtson
University of Iowa
323 EMRB
52242 Iowa City United States
[email protected]
Andreas Koch
BASF SE
Speyerer Strasse 2
67117 Limburgerhof Germany
[email protected]
Stefan Kraiss
Nonnenwald 2
[email protected]
Petr Kralik
Veterinary Research Institute
Hudcova 70
62100 Brno Czech Republic
[email protected]
Koos Kranenborg
BIOKÉ
Plesmanlaan 1d
[email protected]
Jette Dina Kreiberg
Novo Nordisk A/S
Novo Nordisk Park
2760 Måløv Denmark
[email protected]
Vlastimil Krivda
State Veterinary Institute Prague
Sidlistni 136/24
165 03 Prague Czech Republic
[email protected]
Guido Krupp
Fujifilm / AmpTec GmbH
Koenigstrasse 4A
[email protected]
Fabian Kubaty
IDT Biologika GmbH
Am Pharmapark
06861 Dessau-Rosslau Germany
[email protected]
Mikael Kubista
TATAA BIOCENTER
Odinsgatan 18
[email protected]
Thomas Liem
Roche Diagnostics
Hochrütistrasse 39
6005 Luzern Switzerland
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Karsten Lueno
Life Technologies
Frankfurter Strasse 129 B
[email protected]
Gerrit Kuhn
Life Technologies
Baarer Str 78
6300 Zug Switzerland
[email protected]
Antoon Lievens
Institute of Public Health
Rue Juliette Wytsmanstraat 14
1050 Brussel Belgium
[email protected]
Marie-Louise Lunn
Exiqon
Skelstedet 16
DK-2950 Vedbaek Denmark
[email protected]
Jaakko Tuomas Kurkela
Thermofisher
Ratastie 2 (P.O. Box 100)
[email protected]
Kristina Lind
TATAA Biocenter
Odinsgatan 28
411 03 Gothenburg Sweden
[email protected]
Soumya Madhavan
Friedrich-Schiller-University Jena
Neugasse 25
07743 Jena Germany
[email protected]
Elise Labbe
LaSalle Beauvais
60000 beauvais France
[email protected]
Andrea Linkmeyer
TUM - Weihenstephan
Emil-Ramann Str. 2
D-85350 Freising Germany
[email protected]
Rodrigo Manjarin
Michigan State University
1290 Anthony Hall
48823 East Lansing United States
[email protected]
Laura F. Lalonde
Canadian Food Inspection Agency
116 Veterinary Road
S7N 2R3 Saskatoon Canada
[email protected]
Andrea Linnemann
Qiagen GmbH
Qiagenstr. 1
[email protected]
Christian Mann
Thermo Scientific
Im Steingrund 4-6
63303 Dreieich Germany
[email protected]
Olfert Landt
TIB Molbiol
Eresburgstraße 22-23
[email protected]
Ken Livak
Fluidigm Corporation
7000 Shoreline Court, Suite 100
94080 South San Francisco United
States
[email protected]
UGO MARCHESI
ISTITUTO ZOOPROFILATTICO
SPERIMENTALE DELLE REGIONI
LAZIO E TOSCANA
VIA APPIA NUOVA 1411
00178 ROMA Italy
[email protected]
Oliver Latz
Eurofins MWG Operon
Anzinger Str. 7a
[email protected]
Robert Lechner
TU München
Hofmark 1-3
82393 Iffeldorf Germany
[email protected]
Hong Kai Lee
[email protected]
Steve Lefever
University Ghent
De Pintelaan 185
9000 Gent Belgium
[email protected]
Thomas Legrand
GlaxoSmithKline Biologicals
20, rue Fleming, Parc de la Noire Epine
1300 Wavre Belgium
[email protected]
Ursula Leiser
Roche Diagnostics Ltd.
Forrenstrasse
[email protected]
Clarisse Lemaitre
LaSalle Beauvais
[email protected]
Yuk Ming Dennis Lo
The Chinese University of Hong Kong
Department of Chemical Pathology
HK Hong Kong China
[email protected]
Stéphane LOBBENS
38240 MEYLAN France
[email protected]
Robert Loewe
GeneWake GmbH
Forstenrieder Str. 10
[email protected]
Sabine Lohmann
Nonnenwald 2
[email protected]
Katrin Lorenz
Analaytik Jena AG / Biometra GmbH
Konrad-Zuse-Straße 1
07745 Jena Germany
[email protected]
RANIERO LORENZETTI
ISTITUTO ZOOPROFILATTICO
SPERIMENTALE DELLE REGIONI
LAZIO E TOSCANA
VIA APPIA NUOVA 1411
00178 ROMA Italy
[email protected]
Jacek Lubelski
AMT Biopharma
Meibergdreef 61
1105BA Amsterdam Netherlands
[email protected]
Mercedes Marín-Aguilera
Hospital Clínic of Barcelona-Fundación
Clínic
Villarroel 170
[email protected]
Jan Markus
St. Elizabeth Cancer Institute
Heydukova 10
[email protected]
Eva Martinez
Hipra
Avda. La Selva135
17170 Amer Spain
[email protected]
LUZ MARTINEZ AVILES
HOSPITAL DEL MAR
PASSEIG MARITIM
08003 BARCELONA Spain
[email protected]
MARIOS MATARAGAS
Agricultural University of Athens
Iera Odos 75
GR-11855 Athens Greece
[email protected]
Irene Maumnee
KIC
1855 W Taylor St
60612 Chicago United States
[email protected]
Ralf Mauritz
Nonnenwald 2
[email protected]
Irena Mavric-Plesko
Kmetijski Institut Slovenije
Hacquetova ulica 17
SI-1000 Ljubljana Slovenia
[email protected]
Heinrich HD Meyer
Physiology - Weihenstephan
[email protected]
Maren Neef
University of Tuebingen
Auf der Morgenstelle 1
72074 Tübingen Germany
[email protected]
Dietmar Mayer
IDT Biologika GmbH
Am Pharmapark
06861 Dessau Roßlau Germany
[email protected]
Dagmar Michel
Biomed
Bruckmannring 32
85764 Oberchleißheim Germany
[email protected]
Marcus Neusser
Bio-Rad
Heidemannstrasse 169
[email protected]
Nicole Mayländer
Bavarian Nordic GmbH
Fraunhoferstrasse 13
[email protected]
Gabriel Minarik
GENETON Ltd.
Cabanova 14
841 02 Bratislava Slovakia
[email protected]
Anthony Newman
Elsevier
Radarweg 29
1043 NX Amsterdam Netherlands
[email protected]
Axel Moehrle
4titude Ltd.
Sickingenstrasse 26
[email protected]
Bart Nijmeijer
AMT Biopharma
Meibergdreef 61
[email protected]
Peter Molkenthin
InstMikroBioBw
[email protected]
Gerd Nilsen
ArcticZymes AS
Sykehusveien 23
9294 Tromsø Norway
[email protected]
Claire Monk
Primerdesign Ltd
[email protected]
Andreas Nitsche
Robert Koch Institute
Nordufer 20
[email protected]
Shila Mortensen
Statens Serum Institut
Artillerivej
[email protected]
Hans Nitschko
Max von Pettenkofer-Institut
Pettenkoferstr. 9a
[email protected]
Martin Moser
University of Zurich
Winterthurerstrasse 190
8057 Zürich Switzerland
[email protected]
Anna Krestine Noergaard
Danish Technological Institute.
Kongsvang Allé 29 Build. 4
8000 Aarhus Denmark
[email protected]
Ilona Moßbrugger
Institut für Mikrobiologie der Bundeswehr
[email protected]
Mikkel Noerholm
Exosome Diagnostics, GmbH
Am Klopferspitz 19a
[email protected]
Cinthia Moysés
Life Technologies
Av Itacira, 2105
04061002 São Paulp Brazil
[email protected]
Marika Nyman
TUM
AM HOCHANGER 2
85350 FREISING Germany
[email protected]
Jakob Müller
Itzling 6
[email protected]
Sören Ocvirk
BioEPS
Vöttinger Straße 34c
[email protected]
Elke Müller
Genetic ID (Europe) AG
[email protected]
Ralph Oehlmann
IMGM Laboratories GmbH
Lochhamer Str. 29
[email protected]
Alexander Nagy
State Veterinary Institute Prague
Sídlištní 136/24
165 03 Prague 6 Czech Republic
[email protected]
Jakob Ørtvig
AH Diagnostics
Runetoften 18
8210 Aarhus V Denmark
[email protected]
Frank McCaughan
Medical Research Council
MRC Laboratory of Molecular Biology
CB1 3AX Cambridge United Kingdom
[email protected]
Rena McClory
Illumina
9885 Towne Centre Drive
92121 San Diego United States
[email protected]
Johannes Peter Meier
BioEPS
Ludwig-Thoma-Str.23
84307 Eggenfelden Germany
[email protected]
Julia Meiser
Institute for Experimental and Clinical
Pharmacology and Toxicology
Kirrbergerstrasse
66421 Homburg Germany
[email protected]
Jose Melo-Cristino
Faculdade de Medicina da Universidade
de Lisboa
Av Prof Egas Moniz
1649-028 Lisboa Portugal
[email protected]
Lourdes Mengual
Hospital Clinic-Fundació Clínic
Villarroel, 170
[email protected]
ANNA MERCADE
Universitat Autonoma de Barcelona
FACULTAT DE VETERINARIA.
O8193 BELLATERRA Spain
[email protected]
Steffen Mergemeier
CONGEN Biotechnologie GmbH
Robert-Rössle-Str.10
[email protected]
Ssilvia Meschi
Inmi Lazzaro Spallanzani
Via Francesco Carrara 16
55100 Lucca Italy
[email protected]
Swanhild Meyer
[email protected]
Michael Pacher
SunGene GmbH
Corrensstr. 3
06466 Gatersleben Germany
[email protected]
Shiyang Pan
The first affiliated Hospital of Nanjing
Medical University
300, Guangzhou Road, Nanjing, Jiangsu
Province
210029 Nanjing China
[email protected]
Renato Panebianco
University Urbino - Biomolecular
Via I Maggetti, 26/2 (Loc Sasso) 61029
Urbino - Italy
61029 Urbino Italy
[email protected]
Walter Paper
Eppendorf Vertrieb Deutschland GmbH
Johann-Schmidbauer-Str. 4
94351 Feldkirchen Germany
[email protected]
Dario Papi
TIB Molbiol
[email protected]
SPIROS PARAMITHIOTIS
Agricultural University of Athens
Iera Odos 75
GR-11855 Athens Greece
[email protected]
Viresh Patel
[email protected]
Filip Pattyn
Ghent University
De Pintelaan 185
9000 Gent Belgium
[email protected]
Natasha Paul
TriLink BioTechnologies, Inc.
9955 Mesa Rim Road
[email protected]
Michael W Pfaffl
TUM
[email protected]
Paolo Piazza
Wellcome Trust Centre for Human
Genetics
Roosevelt Drive
OX3 7BN Oxford United Kingdom
[email protected]
Paul Pickering
Life Technologies
850 Lincoln Centre Drive, MS 447
[email protected]
Janin Pieritz
Life Technologies
Gutenbergstr.108
70197 Stuttgart Germany
[email protected]
Veronika Pistek
TU München, Weihenstephan
[email protected]
Camille Plusquellec
BIOCORTECH
8 rue Croix de Jarry
75013 Paris France
[email protected]
Michelle Plusquin
Hasselt University
Campus Diepenbeek
3590 Diepenbeek Belgium
[email protected]
Ingo Poleschak
Hauptstrasse 62
85716 Unterschleissheim Germany
[email protected]
Kok-Siong Poon
[email protected]
Sabina Iurian
Pediatric Hospital Sibiu
9-9A Dimitrie Pompeiu street, building
2A, ground floor
[email protected]
Derek Potter
NanoString Technologies
530 Fairview Avenue N
98109 Seattle United States
[email protected]
Sylvia Pfaffl
BioEPS GmbH
Lise-Meitner-Strasse 30
[email protected]
Hervé Pouzoullic
Life Technologies
25, Av de la Baltique B.P. 96
Courtaboeuf
91943 Villebon sur Yvette Cedex France
[email protected]
Matthias Pfeiffer
Eurofins MWG Operon
Anzinger Str. 7a
[email protected]
Rob Powell
PrimerDesign Ltd
SO150DJ Southampton United Kingdom
[email protected]
Henrik Pfundheller
Exiqon
Skelstedet 16
[email protected]
Bukkaraya, S Prakash
NDRI Karnal
NDRI
132001 Karnal India
[email protected]
Sebastian Pünzeler
Adolf-Butenandt-Institut für
Molekularbiologie
Schillerstraße 44
[email protected]
Giorgia Puorro
Università degli Studi di Napoli "Federico
II"
Via Pansini 5, ed. 17
80131 Naples Italy
[email protected]
Alexander Racz
Eurofins MWG Operon
Anzinger Str. 7a
[email protected]
Eugen Radu
Emergency University Hospital
Bucharest
9-9A, Dimitrie Pompeiu Street, building
2A, ground floor
[email protected]
Arjun Raj
University of Pennsylvania
210 S. 33rd St.
19104 Philadelphia United States
[email protected]
Ann Randolph
University of Michigan Health Service
1150 W. Medical Center Dr.
48109 Ann Arbor United States
[email protected]
Sarah Dugdale
GeneSys Ltd
Innovation House
GU16 7PL Camberley United Kingdom
[email protected]
Carolyn Reifsnyder
Agilent Technologies, Inc.
11011 N. Torrey Pines Rd.
92037 La Jolla United States
[email protected]
Rita S. Rein
Nonnenwald 2
[email protected]
Martina Reiter
BioEPS GmbH
Lise-Meitner-Strasse 30
[email protected]
Volker Reuck
TIB Molbiol
[email protected]
Hyoung Seok Rho
Novomics Meditech
250 Seongsanno, Seodaemun-Gu
120-752 Seoul South Korea
[email protected]
Matteo Ricchi
IZSLER
Via Strada Faggiola 1
29027 Gariga di Podenzano Italy
[email protected]
Marianna Romzova
IBT ASCR
Videnska 1083
14200 Prague 4 Czech Republic
[email protected]
Nicolas RICHARD
38240 MEYLAN France
[email protected]
Scott D. Rose
1710 Commercial Park
52241 Coralville United States
[email protected]
Estelle Riche
Merck Millipore
1 rue J. Monod
F-78280 Guyancourt France
[email protected]
Rudi Rossau
Philips
High tech campus 11
5656 AE Eindhoven Netherlands
[email protected]
Heidi Richter
Metabion
Lena-Christ-Straße 44
82152 Planegg- Martinsried Germany
[email protected]
Michael Rosskopf
QIAGEN GmbH
QIAGEN Strasse 1
[email protected]
Stefan Rickenbach
Roche Diagnostics AG
z.H. Antonia Vögel
[email protected]
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Bernhard Roth
Novartis Vaccines & Diagnostics GmbH
Emil-von Behring Str.76
35041 Marburg Germany
[email protected]
Alain RICO
Life Technologies
25 avenue de la baltique
91943 Courtaboeuf France
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Irmgard Riedmaier
TU Munich
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Mary Riley
Lonza
191 Thomaston Street
04841-2994 Rockland United States
[email protected]
Konstantin Rizos
Genetic ID (Europe) AG
[email protected]
Yu-Hui Rogers
J. Craig Venter Institute
9704 Medical Center Drive
20850 Rockville United States
[email protected]
Lorenz Roggo
Roche Diagnostics (Switzerland) Ltd
Industriestrasse 7
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Sameer Rohatgi
Illumina
9885 Towne Centre Drive
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Felix Roll
Biolegio
Lagelandse Weg 56
6545 CG Nijmegen Netherlands
[email protected]
Veronika Rozehnal
Daiichi-Sankyo Europe
Lochhamer Str. 29a
[email protected]
Jan M Ruijter
Academic Medical Center, Amsterdam
Meibergdreef 15
1105AZ Amsterdam Netherlands
[email protected]
Susan Rümmler
Oncoscreen GmbH
Löbstedter Str. 93
07749 Jena Germany
[email protected]
Matevž Rupar
National Institute of Biology
Večna pot 111
[email protected]
Yvet Noordman
AMT Biopharma
Meibergdreef 61
[email protected]
Vendula Rusnakova
Institure of Biotechnology
Videnska 1083
14202 Prague Czech Republic
[email protected]
Ze'ev Russak
Azure PCR
59A Brent Street
[email protected]
Thomas Rygus
Life Technologies
Frühlingsweg 3a
86859 Holzhausen Germany
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Francesco Saccà
Università degli Studi di Napoli "Federico
II"
Via Pansini, 5
80127 Napoli Italy
[email protected]
Angela Sachsenhauser
TU München
[email protected]
Yamuna Sahadevan
Ecology
Emma Heintz strasse 6a
07745 Jena Germany
[email protected]
Roswitha Santner
Life Technologies
Ottenser Marktplatz 8
[email protected]
Ossian Saris
Ratastie 2(P.O.Box 100)
[email protected]
Mihir Sarkar
Indian Veterinary Research Institute
Physiology & Climatology Division
243122 Bareilly India
[email protected]
Christof Schaefauer
genewave
le dorian b2
75011 paris France
[email protected]
Dieter Schams
TUM Weihenstephan
[email protected]
Kimberly Kay Schartl
LONZA Biologics, GmbH
Nattermannallee 1
50829 Köln Germany
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Barbara Schechinger
Illumina
Chesterford Research Park
CB10 1XL Saffron Walden, Essex United
Kingdom
[email protected]
Silvio K. Scheel
QIAGEN GmbH
Konrad-Peutinger-Straße 4
[email protected]
Pia Scheu
Heidemannstrasse 164
80939 Muenchen Germany
[email protected]
Thomas Andreas Schild
Life Technologies
Kantstr. 4
76137 Karlsruhe Germany
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Michael Schleichert
Lambda GmbH
Gewerbepark 2
4261 Rainbach Austria
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Melanie Schneider
Oncoscreen GmbH
Löbstedter Str. 93
07749 Jena Germany
[email protected]
Alfred Schöller
LK Weinviertel Mistelbach
Liechtensteinstrasse 67
2130 Mistelbach Austria
[email protected]
Kati Schroeder
Robert Koch-Institut
Nordufer 20
[email protected]
Rainer Schuster
Amplifa
Rickenbacher Straße 107
88131 Lindau Germany
[email protected]
Rachel Scott
Bio-Rad
[email protected]
Daniela Sebah
LGL
Ringstrasse 28
85764 Oberschleissheim Germany
[email protected]
Eva-Maria Sedlmeier
Gregor-Mendel-Straße 2
[email protected]
Bernhard Setzer
BS-Diagnostik
Waidplatzstrasse 8
79331 Teningen-Nimburg Germany
[email protected]
Amir Houshang Shemirani
Debrecen University
Nagyerdei krt 98
4012 Debrecen Hungary
[email protected]
Francois-Xavier Sicot
Takara Bio Europe
2 avenue Kennedy
F-78100 Saint Germain en Laye France
[email protected]
Anna Siddle
GeneSys Ltd.
Innovation House
GU167PL Camberley United Kingdom
[email protected]
Matthias W. Sieber
University Hospital Jena
Erlanger Allee 101
07743 Jena Germany
[email protected]
Franziska Siegel
Rheinische Friedrich-WilhelmsUniversität Bonn
Sigmund-Freud-Str. 25
53105 Bonn Germany
[email protected]
Tanja Sigl
Technische Universitaet Muenchen
Veitsmuellerweg 4
[email protected]
Carl-Johan Simola
Lonza
Strandhaven 12
2665 Vallensbaek Denmark
[email protected]
Davide Sisti
Via I Maggetti, 26/2
61029 Urbino Italy
[email protected]
Kerstin Skovgaard
Technical University of Denmark
Bülowsvej 27
[email protected]
Iva Slana
Hudcova 70
[email protected]
Michal Slany
Hudcova 70
[email protected]
Virpi Sorri
Thermo Scientific
Keilaranta 16A
02150 Espoo Finland
[email protected]
Ben Sowers
81 Digital Drive
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Mary Span
CYCLERtest
Rotscherweg 61
6374 XW Landgraaf Netherlands
[email protected]
University Hospital Hamburg-Eppendorf
Martinistr. 52
20246 hAMBURG Germany
[email protected]
Wolf D. Splettstoesser
Neuherbergstr. 11
[email protected]
Stefan Springer
FH Weihenstephan
Sanddornweg 6
85375 Neufahrn Germany
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Margrit Stadler
Fluidigm Europe
Parnassustoren
[email protected]
Karen Smeets
Hasselt University
Agoralaan, Building D
3590 Diepenbeek Belgium
[email protected]
Anders Ståhlberg
University of Gothenburg
Cancer Center, University of
Gothenburg, Box 425
[email protected]
Leila Smith
Fluidigm Europe
Parnassustoren
[email protected]
Karin Stangeland
Roche Diagnostics Norge AS
Pb 6610
0607 Oslo Norway
[email protected]
Jan Söderman
Ryhov County Hospital
Länssjukhuset Ryhov
SE-55185 Jönköping Sweden
[email protected]
Michael Steidle
Fujifilm Europe GmbH
Hessenstr. 31
40549 Düsseldorf Germany
[email protected]
Denny Sonnemans
Intervet International bv
Building PP38
5830AA Boxmeer Netherlands
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Julia Steiger
Bioline GmbH
Biotechnologie Park TGZ 2
14943 Luckenwalde Germany
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Ketil Bernt Sorensen
Danish Technological Institute
Kongsvang Alle 29
8000 Aarhus C Denmark
[email protected]
Tanja Stein
Life Technologies
Frankfurter Strasse 129
[email protected]
Diana Sorg
Weihenstephanerberg 3
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Oliver Stephan
Life Technologies
Frankfurter Strasse 129b
[email protected]
Vilberto Stocchi
Via I Maggetti, 26
61029 Urbino Italy
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Jessica Tiedke
University of Hamburg
Martin-Luther King Platz 3
[email protected]
Rob van Miltenburg
DAMP 6164
[email protected]
Martin Thomas Stock
LS für Zoologie WZW TUM
Höhenweg 13
87656 Untergermaringen Germany
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Laura Tizzoni
Cogentech
via Adamello, 16
20139 Milan Italy
[email protected]
Jo Vandesompele
Ghent University & Biogazelle
De Pintelaan 185
9000 Ghent Belgium
[email protected]
Katharina Stöcker
Intervet Innovation GmbH
Zur Propstei
55270 Schwabenheim Germany
[email protected]
Natasa Toplak
Omega d.o.o.
Dolinskova 8
[email protected]
Petra Vasickova
Hudcova 70
[email protected]
Michael Richard Straka
IDEX Health & Science
600 Park Court
94929 Rohnert Park United States
[email protected]
Kirsten Uebel
TU München
[email protected]
David VAUDRY
38240 MEYLAN France
[email protected]
Linda Strömbom
TATAA Biocenter
Odinsgatan 28
41103 Göteborg Sweden
[email protected]
Susanne E. Ulbrich
[email protected]
Markus Veit
Sigma Aldrich
Eschenstr.5
82024 Taufkirchen Germany
[email protected]
Chanchal Sur Chowdhury
University of Basel
University Hospital Basel, Department of
Biomedicine (ZLF)
4031 BASEL Switzerland
[email protected]
Andreas Untergasser
University of Heidelberg
Schlossstr 34
64720 Michelstadt Germany
[email protected]
Enrique Viturro
[email protected]
Irem Uzonur
Fatih University
Buyukcekmece
34500 Istanbul Turkey
[email protected]
Hans von Besser
Eppendorf
Oberer Kühlenberg14
97078 Würzburg Germany
[email protected]
Tomas Szemes
Geneton
Cabanova 14
[email protected]
Agne Valinciute
State Research Institute of Innovative
Medical Center
Bitininku str. 2B-28
08310 Vilnius Lithuania
[email protected]
Tobias Wagner
Life Technologies
Dietramszeller Platz 7
[email protected]
Monika Szyszka-Niagolov
Biosystems Ltd.
2, 6th September Str.
1000 Sofia Bulgaria
[email protected]
Laura Valinotto
Buenos Aires Children's Hospital
Moldes 2969 7A
1429 Buenos Aires Argentina
[email protected]
Jens-Eike Täubert
TU München
Mühlenweg 22
[email protected]
Koenraad van Acker
Biocartis
Antwerpsesteenweg 237
2350 Vosselaar Belgium
[email protected]
Roxana Teisanu
Ecole Polytechnique Federale de
Lausanne
AI 1112 (Bâtiment AI)
CH-1015 Lausanne Switzerland
[email protected]
Pim van der Aar
Dync B.V.
Hortensiastraat 8
4818GM Breda Netherlands
[email protected]
David Svec
TATAA Biocenter AB
Odinsgatan
[email protected]
Gudrun Tellmann
Nonnenwald 2
[email protected]
Ales Tichopad
Academy of Science of Czech Republic
Videnska 1083
14000 Prague Czech Republic
[email protected]
Ronald van Eijk
Leiden University Medical Center
Albinusdreef 2
2300RC Leiden Netherlands
[email protected]
Ruud van der Steen
Biolegio BV
Lagelandse Weg 56
6545 CG Nijmegen Netherlands
[email protected]
Heiko Walch
Roche
Nonnenwald 2
[email protected]
Rudolf Walser
Amplifa
Hattnauerstrasse 18
88142 Wasserburg Germany
[email protected]
Mandy Warnke
Molecular Machines & Industries AG
Flughofstrasse 37
8152 Glattbrugg Switzerland
[email protected]
Barbara Weckerlein
Metabion International AG
Lena-Christ-STr.44
82152 Planegg-Martinsried Germany
[email protected]
Rosemarie Weikard
Leibniz Institute for Farm Animal Biology
Wilhelm-Stahl-Allee 2
18196 Dummerstorf Germany
[email protected]
Karin Tarp Wendt
Technical University of Denmark
Bülowsvej 27
[email protected]
Carsten Winter
Biometra GmbH
Rudolf-Wissell-Straße 30
D-37079 Göttingen Germany
[email protected]
ByoungSu Yoon
Kyonggi University
8114, Dept. Life Science
443-760 Suwon South Korea
[email protected]
Jochen Wettengel
Giggenhauserstr. 1
[email protected]
Vital Wohlgensinger
Microsynth
Schützenstrasse 15
9436 Balgach Switzerland
[email protected]
Yulia Zabiyaka
Mechnikov Research Institute of
Vaccines and Sera, RAMS
First Dubrovskaya st., 15
115088 Moscow Russia
[email protected]
Jim White
NanoString Technologies
21 Acrefield Park
L25 6JX Liverpool United Kingdom
[email protected]
Roman Wölfel
Neuherbergstrasse 11
[email protected]
Jim Wicks
Primerdesign Ltd
[email protected]
Tine Yding Wolff
The Danish Technological Institute
Kongsvang Allé 29
8000 Aarhus Denmark
[email protected]
Steffi Wiedemann
Christian-Albrechts-Universität Kiel
Olshausenstr.
24098 Kiel Germany
[email protected]
Aaron Woodley
Technology Networks Limited
Bull Lane Industrial Estate
CO10 0FD SUDBURY United Kingdom
[email protected]
Marina Wiklander
Sigma-Aldrich
Solkraftsvagen 14C
SE-135 70 Stockholm Sweden
[email protected]
Patricia Susan Wynne
Institut for Pathology
Schönbeinstrasse 40
[email protected]
Anja Mareen Wild
QIAGEN GmbH
Qiagenstr. 1
[email protected]
Michal Wysocki
TUM
Liesel-Beckmann-Straße 1
[email protected]
Wilhelm Windisch
TUM
[email protected]
Remziye YILMAZ
Middle East Technical University
06530 Ankara Turkey
[email protected]
Stephan Zellmeier
Bio-Rad Laboratories GmbH
Heidemannstr. 164
[email protected]
Martin Zevenbergen
Keygene N.V.
Agro Business Park 90
6708 PW Wageningen Netherlands
[email protected]
Philip Zimmermann
ETH Zürich
Universitätsstrasse 2
8092 Zurich Switzerland
[email protected]
Barbara Zschörnig
Jena Bioscience GmbH
Loebstedter Str. 80
07749 Jena Germany
[email protected]
m
© 2011 by Michael W. Pfaffl
qPCR 2011 Event
5th International qPCR Symposium Industrial Exhibition & Application Workshops
Molecular Diagnostics: from single-cells to Next Generation Sequencing
ISBN 9783000338403
Editor:
Michael W. Pfaffl
Physiology, Freising – Weihenstephan
Technical University Munich (TUM)
E-mail
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qPCR 2011 proceedings

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