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 signature based stratification

biomarker signature based stratification

study workflow • meta-analysis of 7 published microarray gene selection of a top expression studies ranking list of 59 • literature screening of almost 800 abstracts from prognostic markers single-gene studies RNA quality control 700 • two PCR-based assays samples • capillary gel electrophoresis (Experion) sample pre-amplification (WT-Ovation) qPCR assay design and • www.rtprimerdb.org validation real-time PCR 384-well plates (LC480) • Prediction Analysis of Microarrays qbase PLUS data-analysis • Kaplan-Meier • Cox proportional hazards

classification of patients with respect to PFS and OS PFS total SIOPEN cohort (n = 312) OS total SIOPEN cohort (n = 313) 100 100 LR n=245 (5) 80 80 survival probability (%) survival probability (%) LR n=245 (42) 60 60 HR n=68 (27) 40 40 HR n=67 (35) 20 20 p = <0.001 (log-rank) p = <0.001 (log-rank) 0 0 50 50 0 100 150 0 100 150 time (months) time (months)

value of the classifier in relation to currently used risk factors: PFS PFS age > 12 months (n = 142) PFS MYCN single copy (n = 265) PFS age <=12 months (n = 172) 100 100 100 survival probability (%) survival probability (%) survival probability (%) 80 80 80 LR n=152 (25) LR n=234 (39) LR n=94 (17) 60 60 60 40 40 40 HR n=48 (24) HR n=31 (18) HR n=20 (12) 20 20 20 p = <0.001 (log-rank) p = <0.001 (log-rank) p = <0.001 (log-rank) 0 0 0 0 50 100 150 0 50 100 150 0 50 100 150 time (months) time (months) time (months) PFS stage 4 (n = 58) PFS not stage 4 (n = 256) PFS MYCN amplification (n = 42 ) 100 100 100 survival probability (%) survival probability (%) survival probability (%) 80 80 80 LR n=222 (31) LR n=8 (1) 60 60 60 LR n=24 (11) HR n=34 (15) 40 40 40 HR n=34 (17) HR n=34 (21) 20 20 20 p = 0.12 (log-rank) p = <0.001 (log-rank) p = 0.22 (log-rank) 0 0 0 60 50 100 150 0 20 40 60 80 100 0 10 20 30 40 50 70 0 time (months) time (months) time (months)

Cox multivariate analysis PAM classifier multivariate cox analysis 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 independent predictor (age, stage, MYCN)

 Vermeulen et al., The Lancet Oncology, 2009

external oligonucleotide standards  synthetic control FP stuffer RCRP  55 nucleotides  PAGE purification  blocking group  5 points dilution series: 150 000 molecules > 15 molecules

external oligonucleotide standards  reproducibility across master mixes (5) and instruments (2) 35 30 25 MM1 20 MM2 MM3 MM4 15 MM5 10 5 0 1000000 100000 10000 1000 100 10

external oligonucleotide standards cross lab comparison 366 samples 5 standards (triplicates) 3 reference genes + 5 genes of interest

external oligonucleotide standards cross lab comparison  5 standards (triplicates) Cq qPCR instrument 1, mastermix 1 36 34 32 average Δ Cq standards 30 28 correction Cq samples 26 24 22 20 18 16 16 18 20 22 24 26 28 30 32 34 36 Cq qPCR instrument 2, mastermix 2

external oligonucleotide standards cross lab comparison  ARHGEF7 gene  366 samples  use of 5 standards (triplicates) for correction Cq 7900HT Cq LC480 abs (dCq)

external oligonucleotide standards cross lab comparison  Vermeulen et al., Nucleic Acids Research, 2009

inter-run calibration requires specialized software  data analysis using qbase PLUS  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 o multiple IRC > more accurate o normalized relative quantities > greater flexibility  dedicated error propagation  automated analysis; no manual interaction required http://www.qbaseplus.com

conclusions  standardization is hot in real-time PCR  MIQE and RDML contribute to higher quality and transparency  external oligonucleotides enable cross-laboratory studies

acknowledgements  Jan Hellemans (UGent, Biogazelle)  Filip Pattyn (UGent)  Steve Lefever (UGent)  Joëlle Vermeulen (UGent)  MIQE & RDML consortium

January 28-29, 2010 Ghent, Belgium www.advances-in-genomics.org early bird registration October 31, 2009