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External oligonucleotide standards enable cross laboratory and - - PowerPoint PPT Presentation
External oligonucleotide standards enable cross laboratory and - - PowerPoint PPT Presentation
External oligonucleotide standards enable cross laboratory and exchange of real-time PCR data Jo Vandesompele professor, Ghent University co-founder and CEO, Biogazelle Roche Molecular Biology Days Vilvoorde, 7 oktober, 2009 biomarker
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biomarker signature based stratification
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- 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 700 samples
- www.rtprimerdb.org
qPCR assay design and validation sample pre-amplification (WT-Ovation) real-time PCR 384-well plates (LC480)
- Prediction Analysis of Microarrays
- Kaplan-Meier
- Cox proportional hazards
qbasePLUS data-analysis
study workflow
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classification of patients with respect to PFS and OS
50 100 150 OS total SIOPEN cohort (n = 313)
survival probability (%)
LR n=245 (5) HR n=68 (27)
p = <0.001 (log-rank)
20 40 60 100 80 time (months) 50 100 150 PFS total SIOPEN cohort (n = 312)
survival probability (%)
LR n=245 (42) HR n=67 (35)
p = <0.001 (log-rank)
20 40 60 100 80 time (months)
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value of the classifier in relation to currently used risk factors: PFS
50 100 150 20 40 60 80 100 10 20 30 40 50 60 70 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)
time (months) 50 100 150 time (months) 50 100 150 time (months) 50 100 150 time (months) time (months) time (months) 100 80 60 40 20
survival probability (%)
100 80 60 40 20
survival probability (%)
100 80 60 40 20
survival probability (%)
100 80 60 40 20
survival probability (%)
100 80 60 40 20
survival probability (%) survival probability (%)
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) MYCN PFS age > 12 months (n = 142) p = 0.22 (log-rank)
100 80 60 40 20
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Cox multivariate analysis independent predictor (age, stage, MYCN) multivariate cox analysis
PAM classifier strong independent predictor: patients with high molecular risk have a 19-fold higher risk to die from disease a 4-fold higher risk for relapse/progression compared to patients with low molecular risk
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Vermeulen et al., The Lancet Oncology, 2009
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synthetic control
55 nucleotides PAGE purification blocking group 5 points dilution series: 150 000 molecules > 15 molecules
RCRP external oligonucleotide standards stuffer FP
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external oligonucleotide standards
reproducibility across master mixes (5) and instruments (2)
5 10 15 20 25 30 35 1000000 100000 10000 1000 100 10 MM1 MM2 MM3 MM4 MM5
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external oligonucleotide standards cross lab comparison
5 standards (triplicates) 3 reference genes + 5 genes of interest 366 samples
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5 standards (triplicates)
external oligonucleotide standards cross lab comparison
average ΔCq standards correction Cq samples Cq qPCR instrument 1, mastermix 1 Cq qPCR instrument 2, mastermix 2
16 18 20 22 24 26 28 30 32 34 36 16 18 20 22 24 26 28 30 32 34 36
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external oligonucleotide standards cross lab comparison
Cq 7900HT Cq LC480 abs (dCq)
ARHGEF7 gene
366 samples use of 5 standards (triplicates) for correction
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external oligonucleotide standards cross lab comparison
Vermeulen et al., Nucleic Acids Research, 2009
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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
- multiple IRC > more accurate
- normalized relative quantities > greater flexibility
dedicated error propagation automated analysis; no manual interaction required
inter-run calibration requires specialized software
http://www.qbaseplus.com
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conclusions
standardization is hot in real-time PCR
MIQE and RDML contribute to higher quality and transparency external oligonucleotides enable cross-laboratory studies
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acknowledgements
Jan Hellemans (UGent, Biogazelle) Filip Pattyn (UGent) Steve Lefever (UGent) Joëlle Vermeulen (UGent) MIQE & RDML consortium
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