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.

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

2. Sample to Insight
IntroductionIntroduction
Agenda
2
Findings within the scientific community
What we have done...
Summary

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

6. Sample to Insight
Introduction
Agenda
6
Findings within the scientific community
What we have done...
Summary

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...”

9. Sample to Insight
Introduction
Agenda
9
Findings within the scientific community
What we have done...
Summary

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!

25. Sample to Insight
Introduction
Agenda
25
Findings within the scientific community
What we have done...
Summary

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?
For up-to-date licensing information and product-specific disclaimers for QIAGEN products,
see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user
manuals are available at www.qiagen.com or can be requested from QIAGEN Technical
Services or your local distributor.