Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?
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Transcript of Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?
Sample to Insight
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Nucleic acid quantification from FFPE samples; are you doing it right?Marion Egli, Global Product Manager, QIAGEN Instruments AG
Nucleic acid quantification from FFPE samples – September 2016
Sample to Insight
Legal Disclaimer
2
QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
For up-to-date licensing information and product-specific disclaimers, 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.
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Outline
The challenges of purifying DNA from FFPE samples1
Variations in DNA quantification results2
Impacts on downstream enzymatic reactions3
Approach for better sample insight4
Nucleic acid quantification from FFPE samples – September 2016
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Facts about FFPE
Current situation FFPE is 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 DNA 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)
Sometimes there is no choice other than FFPE
Nucleic acid quantification from FFPE samples – September 2016
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But FFPE samples are
tricky
Nucleic acid quantification from FFPE samples – September 2016
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Typical challenges with FFPE samples
Effects of formalin fixation on DNA and RNA
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Prolonged fixation severely impacts your sample
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Different factors affect the nucleic acid quality
Sample handling Sample thickness Fixation time Incomplete dehydration Paraffin temperature Sample storage time & temperature Purification conditions
Degradation of nucleic acid
How to overcome these challenges? Learn more at QIAGEN.com/FFPE
Nucleic acid quantification from FFPE samples – September 2016
Sample to Insight
QIAamp DNA FFPE Tissue Kit
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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 20–100 µl 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
Nucleic acid quantification from FFPE samples – September 2016
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Multiple studies investigating variation in FFPE sample processing
And a few open questions:
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...”
Conflicting messagesin studies
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What is the impact of this
and what matters most?
What factors contribute to
variations in DNA quantity?
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Which factor has the highest impact on DNA quantity measured?
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 QIAamp DNA FFPE Kit Automated using the QIAcube or manual
processing With and without RNase digest Assess variability introduced by
purification procedure (manual vs. automated)
DNA quantification Using three different methods,
5 replicates/sample
Assess variability caused by downstream quantification method
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DNA quantification technologies
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
Samples per run 1 Up to 16 1
Drop-and-clean actions required Yes No No
A260/280 Yes Yes
A260/230 Yes Yes
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Comparison of different quantification systems
Concentration variability of purified FFPE samples
variability of concetration determinationInstrument
Nanodrop QIAxpert Qubit
Nuc
leic
Aci
ds (
ng/µ
l)
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
What are the factors contributing to this variability when taking absorbance readings?
QIAxpertNanodrop Qubit A huge variability with all UV/VIS-based
systems
Nanodrop shows the highest variance
Qubit with the lowest variance
Nucleic acid quantification from FFPE samples – September 2016
Nanodrop QIAxpert Qubit
Sample no. 1500 3000 1500
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Does RNase treatment have an influence?
InstrumentNanodrop QIAxpert Qubit
Nuc
leic
Aci
ds (
ng/µ
l)
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
What about automated vs. manual processing?
QIAxpertNanodrop Qubit
Influence on the variance 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
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Does the type of purification have an influence?
Concentration variability with type of purification
InstrumentNanodrop QIAxpert Qubit
Nucl
eic
Acid
s (n
g/µl
)
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
Automated Manual Automated Manual Automated Manual
Kind of Purification
RNAse Digest
NoYes
QIAxpertNanodrop QubitKind of purification
Automated ManualKind of purification
Automated ManualKind of purification
Automated Manual
Higher level of standardization applying
automated sample purification
· with RNase digest· without RNase digest
What role does the tissue type play?
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Comparison of different FFPE tissue types
Concentration variability among different FFPE tissue samples
What role does the FFPE block play?
1. Rat Colon
2. Rat Heart
3. Rat Kidney
4. Rat Liver
5. Rat Muscle
Different FFPE tissue material
leads to different yield of
nucleic acid
Liver tissue is most challenging
because of higher fraction of
RNA
Nucleic acid quantification from FFPE samples – September 2016
QIAxpertNanodrop Qubit
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Contribution to variability in quantification by the block/section
InstrumentNanodrop QIAxpert Qubit
Nucl
eic
Acid
s (n
g/µl
)
0
100
200
300
400
500
600
700
800
1 2 3 1 2 3 1 2 3
FFPE Block
RNAse Digest
NoYes
Higher differences in yields due to the quantification method chosen rather than the block
Nucleic acid quantification from FFPE samples – September 2016
QIAxpertNanodrop Qubit
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What has the greatest influence on quantification of nucleic acids purified from FFPE samples?
ContributionQuantification technology 0.2816Purification method 0.2569Tissue type 0.2288RNase digest 0.1424
FFPE block 0.0542FFPE section 0.0362
The type of quantification technology matters the most!
Nucleic acid quantification from FFPE samples – September 2016
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Why does accurate quantity
and purity assessment
matter?
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Impact of improper quality control on downstream results
Fluorescence readingAbsorbance reading
A method is required that accurately quantifies nucleic acids and detects impurities!
- Sensitivity+ No assay required+ Temperature insensitive+ Detection of contaminants (partly)+ / - Total nucleic acid - No discrimination between DNA and RNA
+ Sensitivity- Assay required - Temperature sensitive+ / - Specific quantification of target- No detection of other NA- No detection of contaminants
Overestimation of DNA concentrationResidual RNA undetected
Weak or no enzymatic reactions
Severe underestimation of DNA concentration due to DNA denaturationResidual other NA or contaminants undetected
Inhibition of enzymatic reactions, false negatives
Nucleic acid quantification from FFPE samples – September 2016
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How to tell what’s really in the
sample?
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DNA quantification technologies
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
Samples per run 1 Up to 16 1
Drop-and-clean actions required Yes No No
A260/280 Yes Yes
A260/230 Yes Yes
Discriminate between molecules of interest No Yes (Yes)
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1. Absorbance measurement (and background correction)
2. Content profiling of the measured spectrum by fitting of reference spectra into:
• Specific DNA or RNA spectrum• Impurities spectrum • Residue spectrum
3. Quality control• Impurities spectrum• Residue spectrum • Background spectrum• A260/A280 ratio for protein
contamination
Spectral content profiling with QIAxpert
RNA SaltsDNAExamples of reference spectra
?unknown
1000 ng/µl total NAvs.
750 ng/µl DNA
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Remember this one?
InstrumentNanodrop QIAxpert Qubit
Nuc
leic
Aci
ds (
ng/µ
l)
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
What would it look like with spectral content profiling?
QIAxpertNanodrop Qubit
Concentration variability of RNase-treated FFPE samples
· with RNase digest· without RNase digest
Nucleic acid quantification from FFPE samples – September 2016
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InstrumentNanodrop QIAxpert Qubit
Nuc
leic
Aci
ds (
ng/µ
l)
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
Results with QIAxpert spectral content profiling
Concentration variability of RNase-treated FFPE samples
QIAxpert A260Nanodrop QubitQIAxpert SCP
· with RNase digest· without RNase digest
Nucleic acid quantification from FFPE samples – September 2016
What is reported by QIAxpert?
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QIAxpert – telling DNA from RNA without a dye
Classic absorbance reading Spectral content profiling
QIAxpert tells you what’s really in the sample
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QIAxpert maximizes the sample insight
Nucleic acid quantification from FFPE samples – September 2016
Quantifying molecule of interest vs. other nucleic acids
Value for DNA
Value for total NA
QIAxpert SCPNanodrop Qubit
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Conclusions
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Summary
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 & spectral content profiling offers reliable quantification of
FFPE samples
QIAGEN provides a number of solutions – from Sample to Insight – supporting your research efforts using FFPE samples
Nucleic acid quantification from FFPE samples – September 2016
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Thank you for your attention!
Questions?
Learn more about our Quality Control Solutions at www.qiagen.com/QCSolutions
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.
Nucleic acid quantification from FFPE samples – September 2016