Extraction of Nucleic Acid From FFPE Tissue Samples

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

Emily ZeringerMarie Gonzalez

March 6th, 2012

Watch this webinar with audio

http://find.lifetechnologies.com/ambion/ffpewebinar/sldshr

2 04/10/23 | Life Technologies™ Proprietary and confidential

Agenda

FFPE Basics

Sample Prep Solutions

Examples of Results

Conclusions



Agenda



FFPE Basics

− What are FFPE samples?

− Why are they important?

− Why are they problematic for molecular biology research?



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….



General Preparation Workflow









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



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



Noticeable differences between FFPE and Unfixed

Unfixed FFPE

Agilent Nanochip Results



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



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

• High Throughput Compatibility:

• Manual Protocols, • Not High Throughput-Compatible



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



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.



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.




Basic Information: qPCR for DNA




Results – In Depth Experiments



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



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.



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



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 samplesTotal RNA Ribo-depleted RNA

FFPE

Unfixed/Frozen



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.



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



Lung small RNA categorical mapping from SOLiD



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



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)

Tumor

spearman = 0.9524 spearman = 0.9579

Normal

spearman = 0.9179 spearman = 0.9342



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)



Agenda



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



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


Extraction of Nucleic Acid From FFPE Tissue Samples

Education