Extraction of Nucleic Acid From FFPE Tissue Samples

Extraction of Nucleic Acid from FFPE tissue samples:
Background, methods, and what to expect

Emily Zeringer
Marie Gonzalez Watch this webinar with audio
March 6th, 2012

Agenda

FPE Basics

ample Prep Solutions

xamples of Results

2 05/29/12 | Life Technologies™ Proprietary and confidential
Watch this webinar with audio

Agenda


FFPE Basics

− What are FFPE samples?

− Why are they important?

− Why are they problematic for molecular biology
research?


What are FFPE samples?
FPE = Formalin Fixed Paraffin Embedded

he most common tissue storage method used today by pathologists for
morphological analysis and diagnosis

issue often appears as the samples shown below:

rocessing and embedding methods are highly variable depending on location and
5
lab but they have a similar workflow…. 05/29/12 | Life Technologies™ Proprietary and confidential

General Preparation Workflow


Why is FFPE Important?

tandard 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

epresents a largely untapped source of samples
− >1 Billion archived FFPE samples, many with clinical annotations

n 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

But…
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


Noticeable differences between FFPE and Unfixed
Agilent Nanochip Results
Unfixed FFPE


Can anything be salvaged?

ith 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


Agenda


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 Watch this webinar with audio

MagMAX™ FFPE Total Nucleic Acid Isolation Kit

elivers faster, cleaner, and easier high-throughput purification of total RNA (including miRNA) and DNA from formalin-
fixed, paraffin-embedded (FFPE) tissue samples.

emove xylene and deparaffinization from your FFPE nucleic acid purifications using novel chemistries

ead-based technology for higher throughput

ue to the nature of FFPE, some application protocols will need to be adjusted to accommodate for best results

igh
15 Throughput Compatibility using Manual and Automated Protocols 05/29/12 | Life Technologies™ Proprietary and confidential

When to use each kit?

ecoverAll
− Small number of samples
− Using larger section thickness
− High-throughput not necessary

agMAX 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.

Agenda


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


Results – Basic Information


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.


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


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


Basic Information: qPCR for DNA

o Sample types
range in Ct
values but all
are within a
usable range


Results – In Depth Experiments


Comparison between formalin-fixed paraffin embedded and
unfixed tissue samples for miRNA Expression between Tumor and
Normal Adjacent Tissue Samples.

urpose:
− Examine the feasibility of using FFPE samples in place of unfixed samples to
analyze differences in miRNA expression between tumor and NAT matched
sets.

ethod
− 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.
25 − Analysis by Raw Ct and Delta Ct between tumor and05/29/12 |samples Proprietary and confidential
NAT this webinar
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Results

Correlation between Raw Ct Values of FFPE and Frozen (unfixed) samples
qRT-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.


Tumor/NAT Delta Ct Values and Correlations
Delta 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

RNA Isolation from fixed and embedded Cell
pellets


Real-time RT-PCR Results


Next-generation sequencing with FFPE Samples


Ribo-depletion from FFPE and Unfixed samples
Total RNA Ribo-depleted RNA

FFPE

Unfixed/
Frozen


Ribo-depletion from FFPE and Unfixed samples
Total RNA Ribo-depleted RNA

FFPE

Unfixed/
Frozen

ibo-Depletion will always be more successful with a fresh/frozen sample vs.
FFPE
− Amplicons are not as compromised, priming rRNA is more successful.

Testing different Platforms for Small RNA

omparing FFPE/unfixed tumor/NAT matched lung samples

OLiD

sed 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

on Torrent Personal Genome Machine (PGM)

sed the Ion Total-RNA Sequencing kit, small RNA protocol.

light advantage with using the PGM for FFPE samples
33 > the speed of sequencing and data analysis allows the user to easily run many technical
05/29/12 | Life Technologies™ Proprietary and confidential

Lung small RNA categorical mapping from SOLiD


Lung small RNA categorical mapping from SOLiD

 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

narrower gel cut range of 60-70 nt instead of 60-80 nt in
current protocol


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.


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)

spearman = 0.9579 spearman = 0.9342
spearman = 0.9524 spearman = 0.9179

Tumor Normal


Between method/tissue concordance - PGM
RP100K = counts per 100k mapped reads (miRBase 16)

s p e ar man = 0. 843 s p e ar man = 0. 840
fresh frozen NAT log2(RP100K)

Observation: source

FFPE NAT log2(RP100K)
of most variation mostly
(and subtly) from tissue
type and not the tissue
Present preparation/storage
miRNAs only method
(row sum
across all runs
>=10 counts)
fresh frozen tumor log2(RP100K) FFPE tumor log2(RP100K)

s p e ar man = 0. 873 s p e ar man = 0. 860
FFPE NAT log2(RP100K)
FFPE tumor log2(RP100K)

fresh frozen tumor log2(RP100K) fresh frozen NAT log2(RP100K)


Agenda


Conclusions

FPE is a difficult sample type
− Caused by both the fixation method and the processing workflow

till 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

roduct solutions available to easily and quickly extract useful
nucleic acid from FFPE samples
− RecoverAll

For Research Use Only. Not for human or animal therapeutic or diagnostic use.


Extraction of Nucleic Acid From FFPE Tissue Samples

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