Challenges of FFPE Sample Materials – Where Does Variation in Quantity of Purified DNA Come From?

Sample to Insight

The challenges of FFPE sample materials – where does variation in quantity of purified DNA come from?

Dr. Carola Schade, QIAGEN GmbH

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IntroductionIntroduction

Agenda

Findings within the scientific community

What we have done...

Summary

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Some facts

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

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Important considerations

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-izationEmbedding 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

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 20–100µl 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

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Introduction

Agenda



Summary

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Multiple studies investigating variation in FFPE sample processing

What impacts downstream results most?

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Some conflicting messages

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...”

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Introduction

Agenda



Summary

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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 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

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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

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)

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DNA – measurement accuracy

173 ng/µl

reference

DNA (Prom

ega 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?

AccuracyReference 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 Sampleno.

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

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DNA – measurement accuracy

5 ng/µl refere

nce 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.

AccuracyReference 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.

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DNA – comparison of linearity using different systems

QIAxpert Nanodrop Qubit

LinearityCalf 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

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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 5-6 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


In total 6000 data points !

w/ RNase digest w/o RNase digest

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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

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

What are the factors contributing to this variability when doing absorbance readings?

QIAxpertNanodrop 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

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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


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

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Does the kind of purification have an influence?

Concentration variability with kind of purification


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

What role does the tissue type play?

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

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Comparison of different FFPE tissue types

Concentration variability among different FFPE tissue samples

Nucleic Acids (ng/µl) vs. Sample Type

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

Rat Colon Rat Heart Rat Kidney Rat Liver Rat Muscle

Sample Type

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


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

Kind of Purification

Different FFPE tissue material leads to different yield of nucleic acids

Liver tissue is most challenging because of higher portion of RNA

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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

Sample to Insight

What is the contribution to the overall variability by the block/section?


21

Less variability when purification is automated


Nuc

leic

Aci

ds (

ng/µ

l)

020406080

100120140160180200220240260280300320340360

Rat C

olon

Rat H

eart

Rat K

idney

Rat L

iver

Rat M

uscle

Rat C

olon

Rat H

eart

Rat K

idney

Rat L

iver

Rat M

uscle

Rat C

olon

Rat H

eart

Rat K

idney

Rat L

iver

Rat M

uscle

Sample Type

RNAse Digest

NoYes

Variability of nucleic acid

concentration determination is

related to different amounts of

RNA

Higher level of standardization

applying automated sample

purification

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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 block or section

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Nucleic Acids (ng/µl) vs. FFPE SampleInstrument

Nanodrop QIAxpert QubitNu

clei

c Ac

ids

(ng/

µl)

0

50

100

150

200

250

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

FFPE Sample

RNAse Digest

NoYes

Contribution to variability in quantification by the block/section

Higher differences in yields due to the quantification method chosen rather than block or section

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What has the highest 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 chosen quantification technology matters most!

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Introduction

Agenda



Summary

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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 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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Q&A session

Thank you for your attention!

Questions?

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Challenges of FFPE Sample Materials – Where Does Variation in Quantity of Purified DNA Come From?