gene-expression studies on limiting sample amounts Stefaan Derveaux - PowerPoint PPT Presentation

RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts Stefaan Derveaux 1,2 , Joëlle Vermeulen 1 1 Ghent University 2 Biogazelle

RNA degradation Small sample Different size platforms Pitfalls and bottlenecks compromising gene expression studies Lack of Inappropriate validation study design Invalidated statistical methods Advances in Genomics, January 28 th 2010

RNA degradation Small sample Different size platforms Pitfalls and bottlenecks compromising gene expression studies Lack of Inappropriate validation study design Invalidated statistical methods Advances in Genomics, January 28 th 2010

RNA pre-amplification 5 to 50 ng RNA RNA pre-amplification WT-Ovation (NuGEN) 5 µg cDNA quantification of ~1000 genes by qPCR Advances in Genomics, January 28 th 2010

RNA pre-amplification 5 to 50 ng RNA 6 neuroblastoma cell lines  4 commercially available  reference RNA samples 740 neuroblastoma  samples RNA pre-amplification WT-Ovation (NuGEN) 5 µg cDNA quantification of ~1000 genes by qPCR Advances in Genomics, January 28 th 2010

pre-amplification yield as a function of RNA input 5 – 15 – 50 ng the yield of pre-amplified cDNA is independent of the amount of input RNA Advances in Genomics, January 28 th 2010

preservation of differential expression after pre-amplification 10 differentially expressed genes dCq = Cq MNS – Cq MNA similar results comparing 6 different cell lines Advances in Genomics, January 28 th 2010

preservation of differential expression after pre-amplification TGFBI Advances in Genomics, January 28 th 2010

correlation of differential gene expression before and after pre-amplification Advances in Genomics, January 28 th 2010

unbiased pre-amplification procedure 194 genes, 4 RNA reference samples, 1164 data points ddCq (bias) dCq NA - dCq A difference in dCq value between 2 samples before and after pre-amplification Advances in Genomics, January 28 th 2010

pre-amplification without introduction of substantial bias lower pre-amplification efficiency the lower the gene is expressed results in higher bias the higher the bias Advances in Genomics, January 28 th 2010

Advances in Genomics, January 28 th 2010

pre-amplification as a function of RNA quality RNA quality control RNA pre- amplification Advances in Genomics, January 28 th 2010

conclusions: linear isothermal Ribo-SPIA pre-amplification method constant yield of pre-amplified cDNA  independent of the amount of input RNA preservation of differential gene-expression  after pre-amplification without introduction of substantial bias generates sufficient material for diagnostic and prognostic work-up  enables large-scale qPCR gene-expression studies using limited amounts of  sample material Advances in Genomics, January 28 th 2010

Acknowledgements  Joëlle Vermeulen  Steve Lefever  Els De Smet  Katleen De Preter  Nurten Yigit  Anne De Paepe http://medgen.ugent.be  Filip Pattyn  Frank Speleman  Jo Vandesompele  Lars Vahlkamp  Bas Hulshof  Roderick Jensen Advances in Genomics, January 28 th 2010