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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
Advances in Genomics, January 28th 2010
gene-expression studies on limiting sample amounts Stefaan Derveaux - - PowerPoint PPT Presentation
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 , Jolle Vermeulen 1 1 Ghent University 2 Biogazelle RNA degradation Small sample Different size platforms Pitfalls
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Advances in Genomics, January 28th 2010
RNA degradation Different platforms Inappropriate study design Invalidated statistical methods Lack of validation Small sample size
Pitfalls and bottlenecks compromising gene expression studies
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Advances in Genomics, January 28th 2010
RNA pre-amplification 5 to 50 ng RNA
RNA pre-amplification WT-Ovation (NuGEN)
5 µg cDNA quantification of ~1000 genes by qPCR
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Advances in Genomics, January 28th 2010
RNA pre-amplification 5 to 50 ng RNA
RNA pre-amplification WT-Ovation (NuGEN)
5 µg cDNA quantification of ~1000 genes by qPCR
- 6 neuroblastoma cell lines
- 4 commercially available
reference RNA samples
- 740 neuroblastoma
samples
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Advances in Genomics, January 28th 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
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Advances in Genomics, January 28th 2010
preservation of differential expression after pre-amplification 10 differentially expressed genes dCq = Cq MNS – Cq MNA similar results comparing 6 different cell lines
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Advances in Genomics, January 28th 2010
preservation of differential expression after pre-amplification
TGFBI
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Advances in Genomics, January 28th 2010
correlation of differential gene expression before and after pre-amplification
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Advances in Genomics, January 28th 2010
unbiased pre-amplification procedure 194 genes, 4 RNA reference samples, 1164 data points ddCq (bias) dCqNA - dCqA difference in dCq value between 2 samples before and after pre-amplification
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Advances in Genomics, January 28th 2010
pre-amplification without introduction of substantial bias
the lower the gene is expressed the higher the bias lower pre-amplification efficiency results in higher bias
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Advances in Genomics, January 28th 2010
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Advances in Genomics, January 28th 2010
pre-amplification as a function of RNA quality
RNA pre- amplification RNA quality control
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Advances in Genomics, January 28th 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
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Advances in Genomics, January 28th 2010
Acknowledgements
- Joëlle Vermeulen
- Steve Lefever
- Els De Smet
- Katleen De Preter
- Nurten Yigit
- Anne De Paepe
- Filip Pattyn
- Frank Speleman
- Jo Vandesompele
- Lars Vahlkamp
- Bas Hulshof
- Roderick Jensen