Scaling PCR Workflows from Benchtop to Automation

Scaling PCR Workflows from
Benchtop to Automation
Scaling PCR Workflows from
Benchtop to Automation
Broadcast Date: Tuesday, June 29, 2010
Time: 1:00 PM EDT
Sponsored by
Scaling PCR Workflows from
Benchtop to Automation
Your Moderator
John Sterling
Editor-in-Chief
Genetic Engineering & Biotechnology News
Sponsored By
Scaling PCR Workflows from
Benchtop to Automation
Lawrence J. Wangh, Ph.D.,
Professor of Biology
Laboratory of Molecular Medicine and Global Health
Brandeis University
Sponsored By
“Sample Prep for Single Cell Single-Tube LATE-PCR”
Lawrence J. Wangh, Ph.D.
Laboratory of Molecular Medicine and Global Health
Department of Biology, Brandeis University, Waltham, MA,
June 29, 2010
Genetic Engineering and Biotechnology News
Webinar
1997 - Small Sample Size
A Challenge to Conventional PCR
Pre-Implantation
Genetic
Diagnosis
Early Cancer
Diagnosis
Limitations of Real-Time PCR
• Low Sensitivity for Small
Number of Initial DNA Targets
CT Value
• No Quantitative End-Point
Analysis!
Freeman et al., (1999) Biotechniques 26: 122-125
A Whole System Approach
Efficient Amplification
of single-stranded DNA
LATE-PCR
PrimeSafe
Sample Preparation
in a single tube
Improved Specificity
PurAmp&
QuantiLyse
Lights On/Lights Off Probes
These New Chemistries Result in New Assay Strategies
RT –LATE-PCR
Dilute’N Go
Sequencing
Multiplexing
Quantitative End-Point
Analysis
see LATE-PCR.org
Uncoupling of
Annealing and
Detection
Virtual Sequencing
Exponential Amplification: 1,2,4,8.16…..
Amplification is Fast, but unreliable
as it slows down.
Therefore measurements are taken
In real-time, as soon as there are
enough molecules
to detect.
real-time
Linear After the Exponential (LATE)
Amplification is fast and is reliably
It switches from exponential to linear at
shortly after the detectable level is
reached. Reduced scatter at end-point
detection background
real-time
detection background
end-point
A Whole System Approach
Efficient Amplification
of single-stranded DNA
LATE-PCR
PrimeSafe
Sample Preparation
in a single tube
Improved Specificity
QuantiLyse
Lights On/Lights Off Probes
These New Chemistries Result in New Assay Strategies
RT –LATE-PCR
Dilute’N Go
Sequencing
Multiplexing
Quantitative End-Point
Analysis
Uncoupling of
Annealing and
Detection
Virtual Sequencing
Pierce, K., Rice, J., Sanchez, J.A., and Wangh, L.J. (2002) “QuantiLyse: Reliable
DNA Amplification from Single Cells”, BioTechniques 32: 1106-1111.
The Problem of Scatter at the
CT Among Single Cells
Pierce et al., (2000). Mol Hum Reprod 6:1155-1164
Optimizing Single-Tube Preparation of Genomic DNA
In Humans
46
Chromosomes
Per
Somatic Cell
Chromosome
Packing Density
1:10,000
XX =
XY =
3 billion base-pairs/Cell
Variations in CT Values Among Replicate Reactions
Reflect Variations in Template Accessibility
44
CT Value
42
39.4
40
 2.58
38
36
35.0
34.4
34
 0.66
 0.34
32
0
10
QuantiLyse
20
30 Denature
40
Heat
in Water
50
60
70
Freeze/Thaw
in Water
80
QuantiLyse: A Simple Reagent and Method for Reliable Preparation of
Genomic DNA in a Single-Tube Reaction
Single Cell, Single Gene, Single Allele
Analysis via Symmetric PCR
-
Normal S ignals
Aberrant S ignals
A Normal/Normal
C Normal/1278
B
D Normal/1278
1900
Fluorescence
-
1278 Signals
1400
900
400
-100
Fluorescence
1900
1278/1278
1400
900
400
-100
11
15
19
23
27
31
35
39
43
Cycle Number
47
51
55
59 11
15
19
23
27
31
35
39
43
47
51
55
59
Cycle Number
Rice et al. (2002), Prenat Diagn 22: 1130-1134
Quality of Genomic DNA Matters!
DNA Shearing Increases Scatter
INTACT GENOMIC DNA: prepared and amplified in the same tube
FROZEN/THAWED GENOMIC DNA
Triplex Reaction 1
Triplex Reaction 2
A Whole System Approach
Efficient Amplification
of single-stranded DNA
LATE-PCR
PrimeSafe
Sample Preparation
in a single tube
Improved Specificity
QuantiLyse
Lights On/Lights Off Probes
These New Chemistries Result in New Assay Strategies
RT –LATE-PCR
Dilute’N Go
Sequencing
Multiplexing
Quantitative End-Point
Analysis
Uncoupling of
Annealing and
Detection
Virtual Sequencing
John Rice, J. Aquiles Sanchez, Kenneth E. Pierce, Arthur H. Reis, Adam Osborne, and
Lawrence J. Wangh (2007) Monoplex/Multiplex Linear-After-The-Exponential (LATE)-PCR
Assays Combined with PrimeSafe® and Dilute-„N‟-Go Sequencing, Nature Protocols 2 #10,
2429-2438.
No PrimeSafe™
Yes PrimeSafe™
3000
3000
10,000 Genomes
2500
2500
1,000 Genomes
Quantitative
End-Point LATE-PCR
10 Genomes
2000
2000
1/25/05
350
3000
3000
300
1500
1500
Fluorescence
TET-Fluorescence ( Rn)
Fluorescence
100 Genomes
250
2500
2500
• Low Scatter Among
Replicates
10 g
Genomes
10010,000
g
Genomes
10001,000
g
100 Genomes
200
1000
1000
• High Sensitivity Even for
Low Numbers of Targets
10 Genomes
2000
2000
150
500
500
100
1500
1500
• Quantitative End-Point
Analysis
50
60
70
50
1000
1000
00
5
20
10
20
30
15
25
30
35
40
40
500
500
-50
Cycle Number
Cycle Number
0
20
30
40
50
60
Cycle Number
70
80
A Whole System Approach
Efficient Amplification
of single-stranded DNA
LATE-PCR
PrimeSafe
Sample Preparation
in a single tube
Improved Specificity
PurAmp&
Lights On/Lights Off Probes
These New Chemistries Result in New Assay Strategies
RT –LATE-PCR
Dilute’N Go
Sequencing
Multiplexing
Quantitative End-Point
Analysis
Uncoupling of
Annealing and
Detection
Virtual Sequencing
Hartshorn C, Anshelevich A, Wangh LJ. (2005) Rapid, single-tube method for
quantitative preparation and analysis of RNA and DNA in samples as small as one
cell. BMC Biotechnol, 5:2.
Separation of the Parts, Rather Than Purification
10,000 kilometers
100,000X
100,000X
The Single-tube PurAmp Method
Xist Gene Expression and Nuclear Localization
in Developing Mouse Embryos
8 – Cell XX
16 – Cell XX
16 – Cell XY
Sheardown et al. Cell 91, 99-107 (1997)
Blastocyst XX
PurAmp is Accurate and Sensitive
Hartshorn et al. (2005) BMC Biotechnology 5:2
Detection of Xist and Sry in Blastocyst Stage Embryos
Hartshorn et al. (2005) BMC Biotechnology 5:2
Laser Zona Drilling for Single Cell Isolation
Does it cause heat shock?
Hartshorn et al. (2003) Mol Reprod Dev 64:41-51
Quantitative Analysis of Hsp 70 Levels
in Single Blastomeres and Whole
Embryos using the PurAmp Method
Hartshorn et al. (2005) Fertility and Sterility 84, no. 5 1547-1550
Summary of Advantages of Single Tube Sample Preparation
• Separation of Components, Rather than Complete Purification
• Experimentally Convenient and Less Expensive
• Quantitatively More Reliable
optimized for minimum scatter among replicates
• Lower Risk of Laboratory Contamination
• Lower Risk of Sample Contamination
• Gentler on substrates, less shear, less degradation
• Fast and Automatable
Summary of Our Synergistic Core Technologies
• LATE-PCR: abundant, reliable, single-strand production
• Dilute’N’Go Sequencing, more convenient, less costly
• Quantitative End-Point Analysis, cheaper, fewer errors
• PrimeSafe, cleaner results and easier multiplexing
• Low Temperature Sequence-Specific Probes
• Low Temperature Mis-match Tolerant Probes
• Lights-On/Lights-Off Probes – high resolution
analysis and “virtual sequencing” in a closed tube
All of the above chemistries are automatable!
Scaling PCR Workflows from
Benchtop to Automation
Gregory L. Shipley, Ph.D.,
Assistant Professor
Director, Quantitative Genomics Laboratory
The University of Texas Health Science Center, Houston
Sponsored By
Scaling Workflows from Bench Top
to Automation
Utilizing Automation for Real-Time qPCR
Gregory L. Shipley, Ph.D.
What do we mean by ‘Automation’?
• Automation refers to using liquid handling robots for
component assembly instead of processing a work flow
manually
• Can be as simple as aspirating and dispensing liquid
from plate A into plate B (or C, D, ... N)
• Can be as complex as automating every step of a
complex workflow utilizing multiple instruments
• Many different kinds of instruments can be integrated
with robot software
A Robotic Workstation - Components
Instruments (l-r)
1-Cytomat 1
2- DTX-880
3- FX- dual arm
96-tip head &
Span-8
4- Cytomat 2
5- ELx405 plate
washer
(not shown)
A Robotic Workstation - Complete
Work Station
inside a
Biosero hepa
filtered hood aseptic
environment
siRNA &
compound
screening with
live cells or
biochemical
assays
Why use automation?
• Automation brings a level of accuracy and precision
to an experiment that can not be achieved manually
for large numbers of repetitions
• For small tasks, 1 or 2 96-well plates, it is faster and
can be just as accurate to do the process manually
• However, using automation means that every plate
will be processed the same, every time regardless of
the number of repetitions, complexity or time
required for the task
What is Involved in using Automation?
• Aside from acquiring the instrumentation, learning to
use the software in a sophisticated way is a critical step
(loops, nested loops, IF statements, variables, etc)
• Requires a dedicated person
• Make sure your assay can be scaled down to 96- or
384-well plates (1536)
• Real-Time qPCR lends itself to this format quite nicely
but not true of all assays
What Liquid Handling Robot to Buy??
• Don’t consider just what you need today, think about the
future
• Make sure the features give you all the flexibility you require
1- How many tips (1, 4, 8, 96, 384) or tools (1 tip or 8 tips)
2- The more tips, the faster the job will be done
3- Span-8 capability = maximum flexibility & speed
4- Using 96 or 384 tip heads are fast but not as flexible work best with another robot
Pipetting with a Liquid Handling Robot
• By default, robot software set to maximize speed but this
minimizes accuracy
• Slow down aspiration & dispense, volume dependent
• Put in delays, 500 - 1500 ms for aspiration - dispense
• Aspirate (2X), dispense- source (1/2X), dispense- target(s)
(1X), dispense remaining- source
• Pre-wet tips if necessary, depends on solution
• Use appropriate size tips/volume
• Use food color dyes for initial program check
• Use tartrazine (10 mM) to check accuracy/absorbance A427
read - A650 background = 5% - 10% CVs
Working with 96-Well Plates
• Originally started setting up 96-well qPCR plates with a
Biomek 2000 in 1996
• Used a single channel tool, one well at a time, due to
asymmetric layout of the RT reactions
• 45 minutes+ to set up one 96-well plate - over an hour to
run the RT reaction on a thermocycler
• Added PCR master mix with the 8-channel tool
• Almost 2 H for real-time qPCR on the ABI 7700
• State of the art at the time
Biomek2000 with Single Channel Tool
Original 96-Well Plate Layout
NAC
Sample
Standard
NTC
ASPrimer
4 μl+6 μl
50 μl PCR
Std 1
Std 2
Std 5
NTC
S #1
S #1
S #1
Std 3
S #7
S #7
S #7
Std 4
S #7
S #14
S #14
S #14
S #14
S #20
S #20
S #20
S #20
S #1
Second Generation 96-Well Plate
Std 1
S #1
Std 2
S #1
S #1
Std 3
S #1
S #8
Std 4
S #8
S #8
Std 5
S #8
NTC
S #15
S #15
S #15
S #15
S #21
S #21
S #21
S #21
Data from the 96-Well Plate
Data from the 96-Well Plate
A Second Assay: 96-Well Plate
Working with 96- & 384-Well Plates
• 1999 shifted to a Tecan Genesis 100, with Span-8
• Span-8 means the tips can expand/contract in the y-axis
and move independently in the z-axis
• Span-8 allows use of up to 8 tips at once vs 1 previously
• Uses fixed tips, no disposable tip costs
• Use 5% Clorox bleach & H2O washes to clean tips
between samples
• Cut the RT setup time to 17 minutes/plate (384)
• Worth every cent
Tecan Genesis 100 with Span-8
Tecan Genesis 100 with Span-8
384-Well Plate- Multiple Sample Layouts
NTC + Standard Curve, 1 Assay/Plate
NTC NTC
NAC
Sample
Standard
NTC
ASPrimer
2 μl+3 μl
20 μl PCR
1
1
5
5
1
1
45 Samples
30 Samples
15 Samples
4
4
3
NAC for each Sample
3
2
2
1
61 Samples
71 Samples
93 Samples
1
93
93
93
93
Two Assays/Plate- 15, 30 or 45 Samples/Half Plate
1 or 2 Sample Sets
NTC + Standard Curve - 2 Assays, 1 plate
15, 30 or 45 Samples, Assay 1, Sample Set 1
NAC for each Sample
15, 30 or 45 Samples, Assay 2, Sample Set 1 or 2
384-Well Plate Data
ABI 7900HT - Human Probe-based CyclinD1 Assay
384-Well Plate Data
ABI 7900HT - Human Probe-based CyclinD1 Assay
Whole-Cell Lysates vs Purified RNA
DMSO vs Staurosporine
• Comparison of making cDNA from whole cell lysates
vs purified RNA
• Treated cells with Staurosporine or DMSO as carrier
• Made cDNA and added to qPCR SYBR master mix
• One 384-well Tox array (Lonza/Bar Harbor Biotech)
per cell culture
• Used 4 cultures for each group
• Total plates is 16
• Loaded plates utilizing a Biomek2000, single
aspirations with quad dispenses, 8-channel tool
Whole-Cell Lysates vs Purified RNA
DMSO vs Staurosporine
Purified RNA
18SrRNA
Cell Lysate
18SrRNA
19 significant
transcript changes
shared between cell
lysates and purified
RNA that validated
with individual
qPCR assays
Automation
essential for this
experiment
Summary
• Choose the robot platform that fits your workflow,
keeping the future in mind
• Become expert at using the software, dedicated user
• Robots are excellent for repetitive, large scale tasks
• Robots are not good if the work flow changes often
• Using the same robot for plasmid or nucleic acid
preps and setting up qPCR is not a good idea
• Always perform QC on new assays before running
real samples
Scaling PCR Workflows from
Benchtop to Automation
David Knorr, Ph.D.,
Applications Manager
Automation Solutions Instruments
Agilent Technologies, Inc.
Sponsored By
Scaling PCR
Automation
David Knorr, Ph.D.
Applications Manager
Agilent Technologies
• Automation rationale, planning and considerations
• PCR workflow
• Different scales of automation
– single liquid handler
– workstation
– integrated system
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Agilent - GEN
29 June 2010
Agilent Technologies
PCR Workflow Solutions
• Instruments for workflow automation
– plate sealer, centrifuge, labeler, stackers, and more
– liquid handlers
› Bravo
› Vertical Pipetting Station (VPrep)
•
•
•
•
56
BenchCel Workstations
BioCel fully automated systems
VWorks software
Reagents
– PCR polymerases (5 choices)
– PCR / qPCR amplification kits
– purification kits for RNA / DNA / PCR product
cleanup
Agilent - GEN
29 June 2010
Automation Rationale
• Capacity or throughput improvements
– shrink research and development timelines
• Quality (product and data) improvements
– process standardization / uniformity
– improve consistency
› reduce error
› reduce subjective data analysis
› enable high-density well formats (e.g. 1536 well plates
• Reduce Operating Costs
• Redistribute brain power - scientists are expensive liquid handlers
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29 June 2010
Plan to Automate
(let the process drive)
• Understand your process;
Scope
Cost
focus on where you’re going and why
• Scope, Timeline and Cost
– hold onto one and the others will fall into place!
• Review:
– current methods
– up / down stream compatibility and bottlenecks
– flexibility
Time
• A bad process = a bad automated process
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29 June 2010
Collaborate with a Good Vendor
• Good components
– ease-of-use, loading, teaching, monitoring, cleaning
– reliable, accessible
– safe
• Good track record
• Good people
– project management
– engineering
– software
– technical support
– service
• Be clear with requirements, communicate often
• Identify a super-user
• Use the system immediately
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Agilent - GEN
29 June 2010
PCR Workflow
Traditional PCR
Real-time PCR
Agilent - GEN
29 June 2010
Nucleic Acid Purification / Isolation
NAPI
• Source material from anything: cells, tissues, fruits, bone fragments, etc.
Each has particular challenges
• Once converted into a liquid (or semi-liquid) nucleic acids can be isolated
usually by some form of affinity chemistry
– magnetic beads
› oligo(dT) or other nucleic acid-binding surface
› requires magnet station & plate-handling robot
– silica-based columns (total nucleic acid  DNase or RNase)
› usually require spinning, or vacuum (robotics)
– ChargeSwitch® technology
• Dedicated automation available
– rarely perform all steps
– formats may not fit remainder of workflow
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Reaction Setup
Setup
Determine process strategy
•
•
•
•
One sample - many reactions
Many samples - ~1 reactions
Layout considerations (tips, reagents, cooling, primers, master mix, etc.)
Applications using similar workflows:
– conventional sequencing
– RNAi library transfection
– magnetic bead purifications
• Capping / sealing
– downstream processing
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PCR Setup: can your head do this?
Setup
• Single tip mode - templates in column 1
1
• Row mode - primers + master mix in row 8
2 • Aliquot master mix - single row of tips
3 • Aliquot templates - single column
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Agilent - GEN
29 June 2010
Agilent PCR Workflow Components
Traditional PCR
Real-Time PCR
Agilent - GEN
29 June 2010
Bravo Handles PCR Workflow
• Small, versatile, lab-friendly footprint
– hood compatible, easy cleaning
• VWorks software
• Gripper robot
• Quick-change pipetting heads
– 96 & 384 disposable & fixed tip
– pin tool (V&P Scientific)
– high accuracy / precision
– 300 nl – 250 µl range
– SBS plates to 1536 wells
– columns / rows
– tube – plate
– tip tracking
• Many accessories
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29 June 2010
Robots Enable Integrated Platforms:
Workstations & Systems
BenchCel
66
Direct Drive Robot
Agilent - GEN
29 June 2010
BenchCel Workstations
• 1 – ~4 instruments
• Low complexity assays or protocols
• Complex arrangements possible
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29 June 2010
WorkStation for PCR setup
•
•
•
•
•
•
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Bravo
BenchCel 6R
BenchCel 4R
PlateLoc
Plate Centrifuge
Multidrop Combi
Agilent - GEN
29 June 2010
BioCel Systems: Maximum Throughput,
BioCel 1200
Hands-Off Automation
• Direct Drive Robot
• VWorks scheduler
– event-driven
– error-handling
– 3rd party drivers
• Environmental control
• Customized protocols
• Limitations:
BioCel 1800
› space
› budget
› imagination
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High-Throughput Genotyping BioCel
• Hardware
– dual enclosures
› 3 liquid handlers (magnetic stations and tip washers)
› seal, X-peel, & spin
› 4°C plate storage
(reagents and primers, 189 plates)
› multiple plate stackers
– 10 thermocyclers
– environmental control
• Protocols
– PCR sample preparation
– RT-PCR clean-up
– sequencing-ready amplicons
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29 June 2010
384,000 PCRs from raw samples? Today?
No problem!
› 5 V11 robots, 1 Staubli robot, 2 translators
› 6 VPreps, 1 Tecan Evo
› 5 Thermo Multidrops, 2 Deerac Equators
› 4°C Liconic tube storage
› BioMicroLab XL9 tube reformatting
› 5 VSpins
› 2 PlateLocs
› 2 VCodes
› 1 computer (VWorks)
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Agilent - GEN
29 June 2010
Scaling PCR Workflows from
Benchtop to Automation
Scaling PCR Workflows from
Benchtop to Automation
Q&A
Sponsored By
Scaling PCR Workflows from
Benchtop to Automation
Thank You For Attending
Scaling PCR Workflows from
Benchtop to Automation
Broadcast Date: Tuesday, June 29, 2010
Time: 1:00 PM EDT
Sponsored by