Pre-amplification critically analysed

Jo Vandesompele

professor, Ghent University

co-founder and CEO, Biogazelle

Advanced Methods in RNA quantification (London, UK)

May 21, 2009

outline

introduction

450 miRNA pre-amplification

Mestdagh et al., Nucleic Acids Research, 2008

[Mestdagh et al., Genome Biology, in press]

whole mRNAome pre-amplification

prognostic gene signature in cancer patients

Vermeulen et al., The Lancet Oncology, accepted

pre-amplification – the one and only quality criterion

preservation of differential expression (fold changes)

before (B) and after (A) sample pre-amplification

[no introduction of bias]

(G1S1)B/(G1S2) B = (G1S1) A/(G1S2) A

G1B/G2B < > G1A/G2A

gene G, sample S, before B, after A

pre-amplification – the scene

pre: before actual qPCR

amplification: make large amounts of RNA/(c)DNA from limited input

single cell – picograms – nanograms >> micrograms

transcriptome wide

all long RNA molecules

advantages

o no prior knowledge of target genes is needed

o study can grow

disadvantages

o somewhat more expensive

focused pre-amplification

predefined set of sequences

advantages

o fast and simple

disadvantages

o all targets need to be known in advance

pre-amplification – the players

transcriptome wide

Eberwine method

o T7-RNA polymerase based in vitro transcription > antisense RNA

o home brew protocols

SMART method (Clontech)

o template switch mechanism sense RNA

o T7-RNA in vitro transcription

Phi29 based

o rolling circle amplification – strand displacement

o cDNA

SPIA technology (NuGEN)

o hybrid RNA/DNA SPIA primer

o cDNA

focused pre-amplification

limited cycle PCR (10-14 cycles)

5’ 3’

5’ 3’

5’

5’3’

AAAAA

UUUUU

UUUUU

AAAAA

AAAAA

TTTTT1 streng cDNA-synthese (met T7-oligo(dT) primer)

ste

1 streng cDNA-synthese(met random hexameren)

ste

2 streng cDNA-synthesede

antisense RNA-amplificatie(T7 RNA polymerase)

TTTTT

TTTTT

5’

3’

3’

5’

3’

5’3’

5’

5’ 3’AAAAA

TTTTT 5’

5’3’

3’

5’UUUUUantisense RNA-amplificatie(T7 RNA polymerase) 3’

2 streng cDNA-synthese(met T7-oligo(dT) primer)

de

NNNNNNNNNNNN

5’ 3’

1 rondeste

2 rondede

Ebermine method

SMART

SPIA

microRNA pre-amplification

stem-loop megaplex reverse transcription using 20 ng total RNA

limited-cycle pre-amplification (14)

qPCR profiling 450 miRNAs and controls

higher sensitivity

minimal amplification bias (Mestdagh et al., Nucleic Acids Research)

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Average CqNP (NBL-S, IMR-32) Average CqNP (NBL-S, IMR-32)∆∆

Cq

(|∆

Cq

NP

-∆

Cq

P|)

NB

L-S

, IM

R-3

2A

ve

rag

e ∆

∆C

q

∆∆

Cq

(|∆

Cq

NP

-∆

Cq

P|)

NB

L-S

, IM

R-3

2A

ve

rag

e ∆

∆C

q

minimal pre-amplification bias

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Cq

-va

lue

Cq

-va

lue

Cq

-va

lue

Cq

-va

lue

total cell number total RNA input (pg)

miR-18a

R2 = 0.975

miR-20b

R2 = 0.993

miR-92

R2 = 0.998

miR-19a

R2 = 0.996

A B

1 2 4 8 16 32 64 128

1 2 4 8 16 32 64 128

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0 2 4 6 8 10 12 14

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single cell profiling

total cell number total RNA input (pg)

Mestdagh et al., Nucleic Acids Research, 2008

outline

background research & goals

neuroblastoma

prognostic marker selection

study design and workflow

RNA quality control

sample pre-amplification

normalization

data-analysis and results

biomarker signature based stratification

biomarker signature based stratification

aim

development and validation of a robust prognostic gene signature for neuroblastoma using real-time qPCR

identifying patients with

increased risk in the current low risk and high risk group

good molecular signature in the current high risk group

better choice of risk-related therapy

neuroblastoma

most frequent extra-cranial solid tumor in children

originates from primitive (immature) sympathetic nervous system cells

1:100,000 children (< 15 years)

20 cases/year Belgium | 700 cases/year USA

15% of childhood cancer deaths

prognosis is dependent on

tumor stage (localized vs. metastatic disease)

age at diagnosis (< or > 1 year)

genetic defects: amplification MYCN, ploidy, loss of 1p, gain of 17q

prognostic classification

misclassifications resulting in overtreatment or undertreatment

need for additional tumor-specific prognostic markers

current microarray gene expression studies

data overfitting

unstable gene lists

lack of overlap

biological & technical noise

much more genes than samples

probe annotation / platform

different risk definition

different data processing and analysis

•meta-analysis of 7 published microarray gene expression studies

• literature screening of almost 800 abstracts from single-gene studies

selection of a top ranking list of 59 prognostic

markers

•two PCR-based assays

•capillary gel electrophoresis (Experion)RNA quality control 423

samples

sample pre-amplification (WT-Ovation)

analysis of 366 primary untreated neuroblastoma tumours using real-time

qPCR

•Prediction Analysis of Microarrays

•Kaplan-Meier

•Cox proportional hazards

data-analysis

study workflow

towards real-time PCR signature profiling

100 ng total RNA

30 ng quality control

10 ng unbiased amplification

WT-Ovation (NuGEN)

PCR assay design and validation

sensitivity, specificity and efficiency

RTPrimerDB

(Pattyn et al., 2006, NAR; Lefever et al, 2009, NAR)

absolute standards

real-time PCR using 384-well format

sample maximization strategy

(Hellemans et al., Genome Biology, 2007)

366 tumors and 1 gene/plate

WT-Ovation reproducibility

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15,00

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35,00

ACTB RPL13A 18S YWHAZ B2M GAPDH UBC HPRT1 SDHA HMBS

me

an

Cq

(n

=3

)

genes

Stratagene

cell line A

cell line B

cell line C

mean of 5, 15 and 50 ng of total RNA amplified

WT-Ovation – no amplification bias

median bias = 0.36, 90%tile bias = 0.61

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cum

ula

tive

dis

trib

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bias (Cq)

WT-Ovation – no amplification bias

median bias = 0.36, 90%tile bias = 0.61

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bias (Cq)

no need for DNase treatment

no need for cleanup of amplified products

preservation of differential expression

qPCR reproducibility

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10 #100 #1,000 #10,000 #

within a 384-well plate: 4 x 96 replicates

qPCR reproducibility

between two identical 384-well plates

maximum ΔCq: 0.45

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synthetic control

55 nucleotides

PAGE purification

blocking group

5 points dilution series: 15 molecules > 150.000 molecules

RCRP

absolute standards

stufferFP

absolute standards

reproducibility across master mixes (5) and instruments (2)

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1000000 100000 10000 1000 100 10

MM1

MM2

MM3

MM4

MM5

absolute standards cross lab comparison

5 standards (triplicates)

5 reference genes + 5

other genes

366 samples

5 standards (triplicates)

absolute standards cross lab comparison

average ΔCq standards

correction Cq samples

Cq

qPCR

instr

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maste

rmix

1

Cq qPCR instrument 2, mastermix 2

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ARHGEF7 gene

366 samples

use of 5 standards (triplicates) for correction

absolute standards cross lab comparison

Cq

qPCR

instr

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ent

1,

maste

rmix

1

Cq qPCR instrument 2, mastermix 2

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SPUD assay (Nolan et al, 2006):

detection of inhibitors

Computed gel analysis (Experion, Biorad):

evaluation of total RNA quality

5’-3’ assay (HPRT1):

evaluation of mRNA integrity

rigorous control of RNA quality

423 primary untreated NB (100 ng total RNA)

30 ng

366 RNA samples

differences in reference gene ranking between intact and degraded RNA

(Perez-Novo et al., Biotechniques, 2005)

impact of RNA quality on expression stability

RNA quality parameters

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RQI 5’-3’ dCq AluSq Cq

frequency

delta-Cq 5’-3’ vs. RQI

RNA samples ordered by average rank (good -> worse)

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RQI

delta-Cq

RNA quality control & sample selection

423 samples:

4 samples < DOT/DOO without event

5 samples < presence of enzymatic inhibitors (SPUD)

20 samples < lack of mRNA integrity (no ΔCq 5’-3’)

o 12/14 failed WT-Ovation

o all low RQI values

28 samples < poor RNA quality (RQI + ΔCq 5’-3’)

366 best samples (86.5 %)

RQI:

o average = 7.4

o median = 7.6

o 90%-tile > 6.1

ΔCq 5’-3’:

o average = 2.36

o median = 2.06

o 90%-tile < 4.75

normalisation using geNorm technology

framework for qPCR gene expression normalisation using the reference gene concept:

quantified errors related to the use of a single reference gene

(> 3 fold in 25% of the cases; > 6 fold in 10% of the cases)

developed a robust algorithm for assessment of expression stability of candidate reference genes

proposed the geometric mean of at least 3 reference genes for accurate and reliable normalisation

Vandesompele et al., Genome Biology, 2002

http://medgen.ugent.be/genorm

geNorm software

automated analysis

ranking of candidate reference genes according to their stability

determination of how many genes are required for reliable normalization

0.003

0.0060.0210.0230.056

NF4

NF1

cancer patients survival curve

statistically more significant results

geNorm validation (I)

log rank statistics

Hoebeeck et al., Int J Cancer, 2006

mRNA haploinsufficiency measurements

accurate assessment of small expression differences

geNorm validation (II)

Hellemans et al., Nature Genetics, 2004

patient / control

3 independent experiments

95% confidence intervals

use of multiple references is now well established

> 1250 citations of our geNorm technology in PubMed

> 8000 geNorm downloads in 100 countries

normalisation using multiple stable reference genes

data analysis using qbasePLUS

based on Ghent University’s geNorm and qBase technology

up to fifty 384-well plates

multiple reference genes for accurate normalization

detection and correction of inter-run variation

dedicated error propagation

automated analysis; no manual interaction required

data analysis

http://www.qbaseplus.com

data analysis using qbasePLUS

based on Ghent University’s geNorm and qBase technology

up to fifty 384-well plates

multiple reference genes for accurate normalization

detection and correction of inter-run variation

dedicated error propagation

automated analysis; no manual interaction required

59 prognostic markers + 5 reference genes

366 samples

hierarchical clustering

survival analysis

Prediction Analysis of Microarrays

Cox proportional hazards modeling

Kaplan-Meier

data analysis

Prediction Analysis of Microarrays

PAM

training test

15 low risk 15 high risk 334 samples

PFS OS

classification of patients with respect to PFS and OS

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OS total SIOPEN cohort (n = 313)

su

rviv

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abili

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%)

LR n=245 (5)

HR n=68 (27)

p = <0.001 (log-rank)0

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time (months)

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PFS total SIOPEN cohort (n = 312)

su

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%) LR n=245 (42)

HR n=67 (35)

p = <0.001 (log-rank)0

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value of the classifier in relation to currently used risk factors: PFS

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LR n=94 (17) LR n=152 (25) LR n=234 (39)

LR n=222 (31)LR n=8 (1)

LR n=24 (11)

HR n=48 (24) HR n=31 (18)

HR n=34 (21)

HR n=34 (15)HR n=34 (17)

HR n=20 (12)

p = <0.001 (log-rank)

p = <0.001 (log-rank)

p = <0.001 (log-rank)p = <0.001 (log-rank)

p = 0.12 (log-rank)

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time (months)

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time (months)

0 50 100 150time (months)time (months)time (months)

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PFS age <=12 months (n = 172)

PFS not stage 4 (n = 256)PFS stage 4 (n = 58)PFS amplification (n = 42 )MYCN

PFS single copy (n = 265)MYCNPFS age > 12 months (n = 142)

p = 0.22 (log-rank)

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Cox multivariate analysis

independentpredictor

(age, stage, MYCN)

multivariatecox analysis

PAM classifier

strong independent predictor:

patients with high molecular risk have

a 19-fold higher risk to die from diseasea 4-fold higher risk for relapse/progression

compared to patients with low molecular risk

RNA quality control cut-off

depends on the application

microarray vs. qPCR

expression difference of the target(s)

abundance & stability of the target(s)

fresh frozen vs. FFPE

based on the performance of our classifier

ROC AUC accuracy analysis

bad good

RQI <3: 0.27 3: 0.82

5’-3’ dCq >7: 0.43 7: 0.79

AluSq Cq >15: 0.13 15: 0.81

conclusions (I)

validation matters – quality control along the entire workflow

assay performance

template quality

normalization

data-analysis

conclusions (II)

largest qPCR gene-expression study (rigourous RNA quality control)

optimized workflow

using minimal amounts of RNA (100 ng)

use of absolute standards (cross-lab comparison)

selected gene list (59) on a large panel of tumours (366 + 223)

robust multigene expression prognostic classifier

validated on an independent set of tumours

independent after controling for other known risk factors

suitable for routine lab tests

this study might form the basis for future research, i.e. prospective studies

cDNA library source for future qPCR gene expression studies

Frank Speleman

Jo Vandesompele

Nadine Van Roy

Katleen De Preter

Jasmien Hoebeeck

Filip Pattyn

Tom Van Maerken

Joëlle Vermeulen

Center for Medical Genetics, Ghent, Belgium

Geneviève Laureys

Gianpaolo Tonini

Olivier Delattre

Jean Bénard

Valérie Combaret

Raymond Stallings

Angelika Eggert

Akira Nakagawara

Matthias Fischer

Grants: Childhood Cancer Fund, Emmanuel van der Schueren foundation, UGent-GOA, FWO, IUAP, IWT

Collaborators

Nurten Yigit

Els De Smet

Liesbeth Vercruysse

Anne De Paepe

acknowledgements