In this slidedeck, we reveal how to get the most from your FFPE samples. We discuss variability in quantity and purity of DNA purified from FFPE samples manually or with automated procedures, assessed by different quantification and quality control methods. You can also learn more about the QIAxpert system and how it can help you gain reliable quantification of FFPE samples.
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Challenges of FFPE Sample Materials – Where Does Variation in Quantity of Purified DNA Come From?
1. Sample to Insight
The challenges of FFPE sample materials – where does
variation in quantity of purified DNA come from?
Dr. Carola Schade, QIAGEN GmbH
3. Sample to Insight
Some facts
3
Trends
FFPE has become a standard method for long-term preservation of tissue biopsies
Large number of unprocessed FFPE samples are archived in tissue banks and
biorepositories
Samples are highly valuable, especially when they are well characterized
Needs
Maximum recovery from precious, small FFPE samples
DNA must be suitable for all types of applications, including NGS
Removal of co-purified RNA (i.e., for DNA sequencing)
Differentiation between artificial and true mutation
Sometimes there is no other choice than FFPE
4. Sample to Insight
Important considerations
4
Sample handling
Time between excision and fixation
Changes in RNA transcript profile and proteins
Formalin fixation
pH of solution, composition of fixative
Thickness of tissue specimen
Duration of fixation process, volume of formalin
solution
Acid-mediated hydrolytic nucleic acid fragmentation
Cytosine deamination
Crosslinking of biomolecules
Sample
handling
Deparaffin-
ization
Embedding StorageFixation Purification
Molecular
analysis
Preparation and archiving Retrieval and analysis
Embedding
Full dehydration
Melting characteristics of paraffin used
Residual water can lead to proteolysis
Sample degradation when using high-melting
temperature paraffin
Storage
Storage temperature
Sample degradation
Challenges when working with FFPE samples
5. Sample to Insight
QIAamp DNA FFPE Tissue Kit
5
Kit specifications
Silica membrane based
Up to 8 sections, each with a thickness of up to
10 µm and a surface area of up to 250 mm2
Purification of genomic DNA & mitochondrial DNA
Elution volume 20–100µl
QIAcube protocol available
Paraffin removal and sample lysis
No need for overnight incubation
Paraffin is dissolved in xylene and removed
Sample lysis under denaturing conditions with
proteinase K (1 h, 56°C)
Incubation at 90°C to reverse formalin crosslinking
Optional RNase treatment step
7. Sample to Insight
Multiple studies investigating variation in FFPE sample processing
7
What impacts downstream results most?
8. Sample to Insight
Some conflicting messages
8
Tissue type(s) not specified
RNase digest Yes/No unclear
Relative yields vs. absolute yields
Systematic deviations in quantification
dependent on method used? “No method
highly superior
to others...”
... it is particularly important to
choose the most reliable and
constant DNA extraction system,
especially when using small
biopsies and low elution volumes...
“ ...variation in pre-PCR steps is
prevalent...”
...all common DNA quantification
techniques can be used for
downstream applications...
“DNA
quantitation may
also impact PCR
efficiency...”
10. Sample to Insight
Which factor has the highest impact on DNA quantity measured?
10
Study design Samples
5 different rat tissue types
2–3 different blocks
5-6x 3 sections, 10 µm thickness
Assess variability caused by the samples
themselves
FFPE Samples
Nanodrop QIAxpert Qubit
In total 6000 data points!
QIAcube Manual
w/ RNase digest w/o RNase digest
DNA purification
Automated using the QIAcube or manual
processing
QIAamp DNA FFPE Kit
With and without RNase digest
Assess variability introduced by operator
(manual vs. automated)
DNA quantification
Using three different methods,
5 replicates/sample
Assess variability caused by downstream
quantification method
11. Sample to Insight
DNA quantification technologies
11
Nanodrop QIAxpert Qubit
Technology
UV/VIS absorbance
reading
UV/VIS absorbance
reading
Fluorescence-based assay
LOD 2 ng/µl (dsDNA) 1.5 ng/µl 10 pg/µl (assay dependent)
Sample volume 1 µl 2 µl 1–20 µl
Measurements needed
for 16 samples
16 1 16
Drop-and-clean actions
required
Yes No No
Reported values
A260 Yes Yes
A280 Yes Yes
A260/280 Yes Yes
A260/230 Yes Yes
Discriminate between
molecules of interest
No Yes (Yes)
12. Sample to Insight
DNA – measurement accuracy
14
173 ng/µl reference DNA (Promega ultra pure gDNA )
All technologies with slight
underquantification
Low %CV value for QIAxpert
(Nanodrop with lower no. of
samples being measured)
Qubit with high mean variation
How was the initial
concentration
determined by
Promega?
Accuracy
Reference DNA (Promega ultra pure gDNA ) was used at a concentration of 173 ng/µL and 5 ng/µl (dilution from original
solution in H2O). A total of 63 replicates were measured on two different QIAxpert systems, 32 replicates were measured on
a Nanodrop 8000, and 15 replicates were additionally measured using the Qubit system.
Instrument Sample
no.
AVG
[ng/µl]
STD
[ng/µl]
%CV
Nanodrop8000 32 167.6 1.7 0.6
QIAxpert 63 163.9 2.4 0.9
Qubit 15 154.9 11.1 4.6
13. Sample to Insight
DNA – measurement accuracy
15
5 ng/µl reference DNA (Promega ultra pure gDNA )
All technologies with slight
underquantification
Mean variation increases for absorbance
reading
Qubit variance seems to be lower,
however, also smaller sample number
that was measured
How was the initial concentration
determined by Promega?
Dilution of original solution may
influence accuracy of
measurement.
Accuracy
Reference DNA (Promega ultra pure gDNA ) was used at a concentration of 173 ng/µL and 5 ng/µl (dilution from original
solution in H2O). A total of 63 replicates were measured on two different QIAxpert systems, 32 replicates were measured on
a Nanodrop 8000, and 15 replicates were additionally measured using the Qubit system.
14. Sample to Insight
DNA – comparison of linearity using different systems
14
QIAxpert Nanodrop Qubit
Linearity
Calf thymus DNA (Life Technolgies) was purified using QIAamp chemistry on the QIAcube, and a serial dilution of the final eluate (137 ng/µl) was generated,
representing 120 ng/µl, 100 ng/µl, 80 ng/µl, 60 ng/µl, 50 ng/µl, 40 ng/µl, and 30 ng/µl. A total of 5 replicates of each dilution were subsequently measured using the
QIAxpert system, a Nanodrop 8000, and the Qubit. Data shown for the QIAxpert reflects total NA measured with the dsDNA QIAamp app.
Nanodrop with a systematic overquantification
Qubit with a systematic underquantification
15. Sample to Insight
Which factor has the highest impact on DNA quantity measured?
15
Study design Samples
5 different rat tissue types
2-3 different blocks
5-6 x 3 sections, 10µm thickness
Assess variability caused by samples
themselves
DNA Purification
Automated using the QIAcube or manual
processing
QIAamp DNA FFPE Kit
w/ and w/o RNase digest
Assess variability introduced by operator
(manual vs. automated)
DNA Quantification
Using three different methods,
5 replicates/sample
Assess variability caused by downstream
quantification method
FFPE Samples
QIAcube Manual
Nanodrop QIAxpert Qubit
In total 6000 data points !
w/ RNase digest w/o RNase digest
16. Sample to Insight
Comparison of different quantification systems
16
Concentration variability of purified FFPE samples
What are the factors contributing to this variability when doing absorbance
readings?
QIAxpertNanodrop Qubit
Nanodrop QIAxpert Qubit
Sample no. 1500 3000 1500
AVG [ng/µl] 66.4 49.4 14.5
STD [ng/µl] 98.8 67.7 10.3
A huge variability with all UV/VIS-based
systems
Nanodrop shows the highest variance
Qubit with the lowest variance
17. Sample to Insight
Does RNase treatment have an influence?
17
What about automated vs. manual processing?
QIAxpertNanodrop Qubit
Nanodrop QIAxpert Qubit
RNase
digest
w/o w/ w/o w/ w/o w/
AVG [ng/µl] 103.2 29.6 82.9 15.7 16.1 13.0
STD [ng/µl] 125.9 31.4 82.3 11.7 11.2 9.1
A huge influence of variation is related to RNA
QIAxpert and Qubit show similar low variance on
RNase-treated samples
Nanodrop shows high variance on RNase-
treated and untreated samples
Concentration variability of RNase-treated FFPE samples
with RNase digest
without RNase digest
18. Sample to Insight
Does the kind of purification have an influence?
18
Concentration variability with kind of purification
What role does the tissue type play?
QIAxpertNanodrop Qubit
Kind of purification
Automated Manual
Kind of purification
Automated Manual
Kind of purification
Automated Manual
Higher level of standardization applying
automated sample purification
with RNase digest
without RNase digest
19. Sample to Insight
Comparison of different FFPE tissue types
19
Concentration variability among different FFPE tissue samples
Rat colon Rat heart Rat kidney Rat liver Rat muscle
Min. conc. [ng/µl] 0 3.6 2.7 0.8 0.3
Max. conc. [ng/µl] 193.3 117.9 252.9 810.3 60.3
STD [ng/µl] 35.4 21.1 71.4 125.0 13.5
Different FFPE tissue material leads to different yield of nucleic acids
Liver tissue is most challenging because of higher portion of RNA
20. Sample to Insight
20
Comparison of different FFPE tissue types
Concentration variability among different FFPE tissue types
Variability of nucleic acid
concentration determination is
related to different amounts of
RNA
QIAxpert and Qubit show
similar low variance on RNase-
treated samples
Nanodrop shows high variance
on RNase-treated and
untreated samples
Main contriubtion to variability related to RNA amounts
21. Sample to Insight
What is the contribution to the overall variability by the block/section?
Comparison of different FFPE tissue types
21
Less variability when purification is automated
Variability of nucleic acid
concentration determination is
related to different amounts of
RNA
Higher level of standardization
applying automated sample
purification
22. Sample to Insight
Contribution to variability in quantification by the block/section
22
Higher differences in yields due to the quantification method chosen
rather than block or section
23. Sample to Insight
Contribution to variability in quantification by the block/section
23
Higher differences in yields due to the quantification method chosen
rather than block or section
24. Sample to Insight
What has the highest influence on quantification of nucleic acids purified
from FFPE samples?
24
%Contribution
Quantification technology 0.2816
Purification method 0.2569
Tissue type 0.2288
RNase digest 0.1424
FFPE block 0.0542
FFPE section 0.0362
The chosen quantification technology matters most!
26. Sample to Insight
Summary
26
FFPE tissue samples present a number of challenges
If you really want to be sure that the genomic DNA you quantify represents what
is in your sample:
Choose your quantification technology carefully
Automate your sample prep (i.e., using a QIAcube)
Apply a RNase digestion step
Be aware of systematic differences between technologies when quantifying
nucleic acids
QIAxpert system offers reliable quantification of FFPE samples
QIAGEN provides a number of solutions – from Sample to Insight –
supporting your research efforts using FFPE samples
27. Sample to Insight
Q&A session
27
Thank you for your attention!
Questions?
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