Extraction of Nucleic Acid from FFPE tissue samples: Background, methods, and what to expect
Emily ZeringerMarie Gonzalez
March 6th, 2012
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Agenda
FFPE Basics
Sample Prep Solutions
Examples of Results
Conclusions
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Agenda
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FFPE Basics
− What are FFPE samples?
− Why are they important?
− Why are they problematic for molecular biology research?
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What are FFPE samples? FFPE = Formalin Fixed Paraffin Embedded
The most common tissue storage method used today by pathologists for morphological analysis and diagnosis
Tissue often appears as the samples shown below:
Processing and embedding methods are highly variable depending on location and lab but they have a similar workflow….
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General Preparation Workflow
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General Preparation Workflow
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General Preparation Workflow
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Why is FFPE Important?
Standard histology practice used for over fifty years to maintain tissue structure and prevent putrefaction of tissue− Still the most common tissue storage method used today
Represents a largely untapped source of samples − >1 Billion archived FFPE samples, many with clinical annotations
In the last decade recovery and analysis of both DNA and RNA from FFPE has become an increasingly valuable research tool. − Central to cancer and disease research, biomarker discovery and personalized
medicine
− Target use with: qRT-PCR, microarrays, methylation studies, miRNA work, sequencing
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But…M
any challenges to nucleic acid (NA) Purification from FFPE tissue is due to nature of the processing and storage
1) NA contains “modifications” which can interfere in downstream applications
o Formaldehyde is a promiscuous cross-linker that reacts with proteins, creating a tightly-locked three-dimensional network that also cross-links to other macromolecules (including nucleic acid).
o Some of these cross-links can remain between the nucleic acid and small peptides after purification (as “modifications”) potentially affecting reactions down-stream
2) NA from FFPE is usually degraded/fragmented to varying degrees
o Standard processing/embedding protocols require heating the formaldehyde-soaked samples which can cause degradation
o No standardized processing/embedding workflow between institutions
o the NA in tissue blocks can degrade during standard storage although the chemistry behind this is unclear
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Noticeable differences between FFPE and Unfixed
Unfixed FFPE
Agilent Nanochip Results
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Can anything be salvaged?
With a few modifications, RNA and DNA from FFPE samples can be used successfully in downstream applications:− Use smaller amplicon sizes for qRT-PCR to account for fragmentation and
modification
− If possible, use a larger input of mass (based on spectrophotometer analysis) to account for less usable template
− Adjust protocols for use with small inputs of material
− Use an extraction method that maximizes recovery of all sizes of NA
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Agenda
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RecoverAll™ Total Nucleic Acid Isolation Kit
• Optimized for isolation of total nucleic acids, including microRNAs, from FFPE tissue
• Isolation Technology:
• Spin Column (Glass Fiber Filter) • De-paraffinization steps included• On-filter nuclease treatment• 30 min Protease digestion for RNA • Overnight protease digestion for DNA
• Downstream applications:
• Suitable for real-time RT-PCR and PCR, end-point PCR, mutation screening, northern blotting, miRNA analysis, sequencing, and microarray analyses
• Due to the nature of FFPE, some application protocols will need to be adjusted to accommodate for best results
• Starting Material (Amount):
• Up to four 20 µm sections, up to 35 mg of un-sectioned core samples
• High Throughput Compatibility:
• Manual Protocols, • Not High Throughput-Compatible
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MagMAX™ FFPE Total Nucleic Acid Isolation Kit
Delivers faster, cleaner, and easier high-throughput purification of total RNA (including miRNA) and DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples.
Remove xylene and deparaffinization from your FFPE nucleic acid purifications using novel chemistries
Bead-based technology for higher throughput
Due to the nature of FFPE, some application protocols will need to be adjusted to accommodate for best results
High Throughput Compatibility using Manual and Automated Protocols
> Available pre-written for the MagMAX™ Express-96 Deep Well Magnetic Particle Processor
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When to use each kit?
RecoverAll− Small number of samples
− Using larger section thickness
− High-throughput not necessary
MagMAX FFPE− Larger number of samples
> Greater than 12
− High-throughput (manual or automated) needed
− Organic solvents (such as xylene) aren’t available or can’t be used.
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Agenda
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Examples of Results
− Basic information from different tissue types for RNA and DNA
> Yield
> Real-time RT-PCR and PCR
− mRNA, miRNA, and DNA
− In-Depth Experiments
> Tumor/NAT comparison between FFPE and unfixed matched tissue
> Results from FFPE cell pellets
> Next Generation Sequencing
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Results – Basic Information
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Basic Information - Yield
o The tables below list average yield for both RNA and DNA from different tissues isolated from both kits.
o Yield (ug nucleic acid) can be highly variable between different tissue types, different blocks, and different sections cut from different parts of the block.
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Basic Information: qRT-PCR for mRNA
o Amplicon sizes for each target are less than 100bp to compensate for degradation and modifications
o Sample types range in Ct values but all are within a usable range
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Basic Information: qRT-PCR for miRNA
o FFPE samples seem to work better for miRNA analysis due to the small size of the RNA.
o Sample types range in Ct values but all are within a usable range
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Basic Information: qPCR for DNA
o Sample types range in Ct values but all are within a usable range
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Results – In Depth Experiments
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Comparison between formalin-fixed paraffin embedded and unfixed tissue samples for miRNA Expression between Tumor and Normal Adjacent Tissue Samples.
Purpose: − Examine the feasibility of using FFPE samples in place of unfixed samples to
analyze differences in miRNA expression between tumor and NAT matched sets.
Method− Total RNA Isolation from matched FFPE/Frozen and tumor/NAT sets of lung and
breast tissue samples using the Ambion mirVana™ miRNA Isolation Kit for the frozen samples and Ambion’s RecoverAll™ Total Nucleic Acid Isolation Kit for the FFPE samples
− Downstream analysis for miRNA with 2 step qRT-PCR using a panel of 12miRNA targets.
− Analysis by Raw Ct and Delta Ct between tumor and NAT samples
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Results
Correlation between Raw Ct Values of FFPE and Frozen (unfixed) samplesqRT-PCR reactions were run, in triplicate, for tumor and NAT RNA samples isolated from matched Frozen and FFPE breast and lung tissue from four separate patients. Raw Ct values were calculated then graphed against each other for FFPE and Frozen samples.
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Tumor/NAT Delta Ct Values and CorrelationsDelta Ct values were calculated for tumor and NAT samples for each matched Frozen and FFPE breast and lung tissue sample for four individuals.- Panel A shows the correlation between the FFPE and frozen samples over all four individuals for each tissue type- Panel B shows the delta Ct values for one individual from each tissue type
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RNA Isolation from fixed and embedded Cell pellets
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Real-time RT-PCR Results
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Next-generation sequencing with FFPE Samples
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Ribo-depletion from FFPE and Unfixed samplesTotal RNA Ribo-depleted RNA
FFPE
Unfixed/Frozen
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Ribo-depletion from FFPE and Unfixed samplesTotal RNA Ribo-depleted RNA
FFPE
Unfixed/Frozen
Ribo-Depletion will always be more successful with a fresh/frozen sample vs. FFPE− Amplicons are not as compromised, priming rRNA is more successful.
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Testing different Platforms for Small RNA Comparing FFPE/unfixed tumor/NAT matched lung samples
SOLiD
− Used SOLiD Total RNA-Sequencing kit, small RNA protocol, to create libraries from the matched samples
> Standard protocol followed for all samples except tested narrower gel cuts during gel size selection for FFPE samples to see if provided any advantage
Ion Torrent Personal Genome Machine (PGM)
− Used the Ion Total-RNA Sequencing kit, small RNA protocol.
− Slight advantage with using the PGM for FFPE samples
> the speed of sequencing and data analysis allows the user to easily run many technical replicates for a library
> Useful with the low library input common to FFPE samples
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Lung small RNA categorical mapping from SOLiD
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Lung small RNA categorical mapping from SOLiD
narrower gel cut range of 60-70 nt instead of 60-80 nt in current protocol
Narrower gel cut decreases filtered reads (tRNA) by ~5% while other mapped categories are largely unchanged; slightly more uniquely mapped for the narrow cut
FFPE tumor RNA has 5-10% more total reads mapped with fewer filter matches
Fresh frozen NAT has the least unmapped and filtered reads
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Mapping statistics
Observation: FFPE samples have fewer reads aligning presumably due to RNA quality or effects of formaldehyde on nucleic acids. This has been observed in experiments using SOLiD
Degraded tRNA and rRNA makes it harder to size select the library.
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STaR-Seq SOLiD data from 11/2010: pair-wise comparisons between log2(RPMmiRBase) FFPE and fresh frozen on matched lung samples
x=FFPE (60-70 nt), y=FFPE (60-80 nt)x=FFPE, y=Frozen (60-70 nt)
Tumor
spearman = 0.9524 spearman = 0.9579
Normal
spearman = 0.9179 spearman = 0.9342
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Between method/tissue concordance - PGM
fresh frozen tumor log2(RP100K)
RP100K = counts per 100k mapped reads (miRBase 16)
fres
h fr
ozen
NAT
log2
(RP1
00K)
spearman= 0.843
FFPE tumor log2(RP100K)
FFPE
NAT
log2
(RP1
00K)
spearman= 0.840
spearman= 0.873
FFPE
tum
or lo
g2(R
P100
K)
fresh frozen tumor log2(RP100K)
spearman= 0.860
fresh frozen NAT log2(RP100K)
FFPE
NAT
log2
(RP1
00K)
Observation: source of most variation mostly (and subtly) from tissue type and not the tissue preparation/storage method
Present miRNAs only (row sum across all runs >=10 counts)
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Agenda
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Conclusions
FFPE is a difficult sample type− Caused by both the fixation method and the processing workflow
Still possible to use nucleic acid from FFPE samples in downstream analysis applications− Can Successfully perform real-time RT-PCR and small RNA sequencing
− Does require a few protocol tweaks to compensate for modifications and degradation
Product solutions available to easily and quickly extract useful nucleic acid from FFPE samples− RecoverAll
− MagMax FFPE
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For Research Use Only. Not for human or animal therapeutic or diagnostic use.
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