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

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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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Sample to Insight

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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


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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


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But FFPE samples are

tricky


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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


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


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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


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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https://www.google.ch/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiSqeymqofPAhWGKiwKHcNOAa4QjRwIBQ&url=https://www.qiagen.com/gb/resources/download.aspx?id%3De41041ee-99a7-404a-9494-08f07e3afed3%26lang%3Den&bvm=bv.132479545,d.bGg&psig=AFQjCNFBqPRh-8PepXqO_dpY6unp3O-LjQ&ust=1473682403488478


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Comparison of different quantification systems

Concentration variability of purified FFPE samples

variability of concetration determinationInstrument


Nuc

leic

Aci

ds (

ng/µ

l)

0

50

100

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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



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

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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


Nucl

eic

Acid

s (n

g/µl

)

0

50

100

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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


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



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Contribution to variability in quantification by the block/section


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



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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!


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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


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How to tell what’s really in the

sample?


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DNA quantification technologies


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)


http://www.google.ch/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiimIDhpIfPAhVH_SwKHZrqApQQjRwIBw&url=http://ase.tufts.edu/chemistry/walt/sepa/loaning.html&psig=AFQjCNGhGia2pczQPvkcS739lXAUHA2K4g&ust=1473682329774282



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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?


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What would it look like with spectral content profiling?





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Nuc

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Results with QIAxpert spectral content profiling


QIAxpert A260Nanodrop QubitQIAxpert SCP



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


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


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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.


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Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?