View poster

Localized gene expression changes by AmpliSeq transcriptome sequencing
from Arcturus™ laser capture microdissected formalin-fixed, paraffinembedded (FFPE) Alzheimers and normal brain sections
Stephen M Jackson and Kamini Varma, Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080
Global gene expression analysis of regions of normal and Alzheimer brain
ABSTRACT
We have developed a simplified workflow that enables unbiased
transcriptome analysis of specifically selected cells from fresh frozen or
FFPE archived brain samples. Archival frozen sections and FFPE
sections of temporal lobes from Alzheimer-affected and normal brains
were obtained from a commercial source. Regions of brain tissue were
collected using an ArcturusXT system and RNA eluted from these was
sequenced for gene expression of the transcriptome (~20,000 genes).
LCM allowed us to detect differences in expression patterns in
morphologically distinct regions of the brain as well as detect patterns of
expression related to the Alzheimer pathology iin the immediate vicinity of
pathogenic plaques and tangles.
Finally, we show that using LCM to enrich cells around the plaques and
tangles enabled the detection of gene expression changes that were
undetectable in whole tissue scrapes.
This workflow will enable
researchers to gain novel insights into questions requiring analysis of gene
expression patterns of extremely discrete cell populations in an otherwise
heterogeneous tissue source.
Isolation
Sample
Library Prep
Arcturus Laser Arcturus Paradise
Ion AmpliSeq
Capture
Plus RNA
Whole
Microscopy
extraction
transcriptome kit
Sequencing
Data Analysis
Torrent Suite
Software
AmpliSeq RNA
plug ins
Ion S5 or
Ion Proton
Annotation
GO-term
pathway
analysis
Figure 1. LCM to Ampliseq Transcriptome workflow. The Arcturus product line offers a dissecting microscope using a proprietary infrared laser for tissue capture, as well as reagent kits
optimized for RNA recovery from small amounts of captured cells. The Ion AmpliSeq Transcriptome Human Gene Expression solution facilitates NGS library construction from small
amounts of RNA using a proprietary, ultrahigh-multiplexed PCR amplification approach. The Ion Chef™ System (not shown) automates templating and loading of the prepared library onto a
semiconductor chip and prepares it for sequencing. The Ion S5™ Systems and the Ion Proton™ System can generate up to 80 million reads on a single chip. Finally, the Torrent Suite™
Software facilitates the alignments and analysis of the sequence data.
INTRODUCTION
Samples captured by LCM from morphologically similar normal and Alzheimers brain regions
showed consistent patterns of gene expression (Figure 2).
In our study, we utilized tissues collected from Alzheimer and normal
brains to investigate gene expression differences between small amounts
of discrete neural tissue. By gaining access to extremely small amounts of
tissue using this new workflow and tools, we have gained insights into
gene disregulation underlying Alzheimers disease etiology. Beyond this
specific study, the workflow promises to help researchers gain novel
insights into broader cellular questions that might require gene expression
analysis of of extremely discrete cell populations from an otherwise
heterogeneous tissue source.
A
B
Figure 5. LCM detects changes that would be missed in bulk tissue
analyses. In Alzheimer and normal whole tissue-scrape (WTS) samples,
minimal gene expression differences are seen in sub-regions captured by
LCM, from Alzheimer and normal plaque and tangle samples (P&T), lgene
expression differences are more easily resolved.
Note, some genes have no or low detectable expression in whole tissue
scrapes, but are differentially expressed in the LCM-collected plaque and
tangle samples (red arrows).
Alzh1
Alzh2
Alzh3
Norm1
Norm2
Norm3
1
2
We have developed a simplified workflow that begins with isolation of
specifically selected cells from fresh frozen or FFPE archived brain
samples using the Arcturus XT LCM System and enables unbiased
transcriptome analysis using the Ion Torrent next generation sequencing
(NGS) system.
3
Investigators have long been archiving dissected brain or other neural
samples in tissue blocks and microscopic slides. These typically are
either fresh samples, preserved by freezing and storing in cryroprotective
media, or formalin fixed and embedded in paraffin (FFPE). Due to its ease
of implementation and histological stability, a majority of these samples
have been preserved as FFPE tissues. A significant drawback of using
tissues fixed in this manner is the molecular crosslinks created by formalin
used to preserve the tissue. Prior to analysis using molecular techniques,
such as DNA sequencing, these crosslinks must be reversed, often
resulting in degradation of the DNA.
Despite these challenges,
retrospective analysis of FFPE samples remains very attractive due to
their associated rich clinical data.
Portion of 20,000 gene heat map, showing 500 genes with greatest difference and highest average expression
Subset of transcriptome data, selected for greatest difference between
layers and highest levels of expression (200 genes)
Understanding the development of the brain, the normal and abnormal
function in diseased states requires investigators to analyze the different,
complex substructures of nervous tissue.
The use of unbiased
approaches to researching the function of these components are
particularly appealing given the lack of information currently available on
the role played by these, in many cases, microscopic substructures. With
this in mind, whole transcriptome analysis of extremely small populations
of cells promises to reveal new molecular insights into brain function.
RESULTS
Reproducibility of protocol
C
C
Figure 3. LCM transcriptome analysis of frozen brain samples show good reproducibility. (A)
Heat map of the expression level of 200 high-expressing genes with the greatest difference between the
three layers captured shown in Figure 2. Clear differences in expression patterns can be seen,
suggesting regional specialization. (B) Three separate 2000µ2 circles were captured from similar
regions of a normal and an Alzheimer temporal lobe. A portion of the transcriptome is represented in the
heat map, focusing on the 500 genes with highest average and greatest difference in expression. Note
that the patterns of expression within the two sets of triplicates correlate very well. (C) GO-term
pathway analysis of genes that are differentially represented in the Alzheimer vs normal samples.
A
B
D
MATERIALS AND METHODS
Frozen and FFPE-preserved human brain tissue sections on charged glass slides were
purchased from a commercial source (Biochain, Fremont CA). Slides were prepared for
LCM by rehydration through an ethanol series, staining with Arcturus Paradise Staining
reagent, dehydrated through an ethanol series. Dissected samples were captured onto
macrocaps using an ArcturusXT system. Total RNA was isolated from LCM-captured
cells using the Arcturus Picopure RNA extraction kit, according to the protocol provided in
the kit. Ion AmpliSeq Transcriptome libraries were prepared using the maximal amount of
RNA eluted from the LCM samples (7µl). The standard Ion AmpliSeq library construction
protocol was followed using the Ion AmpliSeq Transcriptome Solution reagents and the
following modifications: cDNA synthesis time was increased to one hour, the number of
Ion AmpliSeq PCR cycles was increased to 30, and the number of final library PCR
amplification cycles was increased to 10. The amplified libraries were templated onto Ion
PI Chips using the Ion Chef System, and sequenced on Ion Proton sequencers. Reads
were aligned to the human transcriptome based on hg19 and normalized to Reads per
Million (RPM) using Torrent Suite Software and ampliseqRNA plug-in embedded in the
software.
A
B
Figure 4. Changes in gene expression levels in Alzheimer vs
normal samples are consistent between FFPE and frozen
samples. Fold changes in 400 high-expressing gene showing
greater than 2-fold expression correlate well in the 2000µ sections
FFPE- and frozen collections. Note that the normal and Alzheimer,
frozen and FFPE samples were from four different donors.
C
3
CONCLUSIONS
2
1
Before dissection
“Grey matter”
Figure 6. LCM is useful for examining gene abundance
differences around pathogenic structures. Clustered heat
map illustrating the genes overrepresented (A) and
underrepresented (B) in the Alzheimer sample. AlzhP&T 1
and NormP&T 1 indicate samples collected from 200µ2 circles;
AlzhP&T 2 and NormP&T 2 are from the 100µ2 samples.
C. Portion of Alzheimer FFPE sample, showing presumptive
plaques and tangles (arrows). One hundred 200µ circles, or
two hundred 100µ circles, were collected around these
anomalies. D. Image of tissue left after collection (left) and
the tissue collected on the macrocap (right). Similar sized
collections were made from normal tissue.
After dissection
“White matter”
Figure 2. LCM based capture of different regions of the brain. A. Image of temporal lobe
of Alzheimer brain, with the areas enriched in white matter and grey matter indicated. B and
C. Three regions microdissected are shown before LCM (B) and left after microdissection (C).
• Arcturus™ XT™ LCM system enables investigators to isolate and analyze unique groups
of cells in brain tissue
• Arcturus LCM coupled with Ion Torrent NGS can be used to analyze molecular differences
at subcellular resolution
• Arcturus LCM facilitates the enrichment of diseased tissue away from unaffected cellular
background, revealing gene expression differences that would be missed from macrodissected tissue
• Ion Ampliseq™ Transcriptome Human Gene Expression Research Panel enables
scientists to identify gene expression changes from extremely limited amounts of difficult
tissue preparations
For Research Use Only. Not for use in diagnostic procedures.
© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks
are the property of Thermo Fisher Scientific and its subsidiaries unless
otherwise specified.