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RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts

Joëlle Vermeulen1, Stefaan Derveaux1, Steve Lefever1, Els De Smet1, Katleen De Preter1, Nurten Yigit1, Anne De Paepe1, Filip Pattyn1, Frank Speleman1 and Jo Vandesompele12*

Author affiliations

1 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium

2 Biogazelle, Ghent, Belgium

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Citation and License

BMC Research Notes 2009, 2:235  doi:10.1186/1756-0500-2-235

Published: 25 November 2009

Abstract

Background

The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR.

Findings

The linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression of 20 genes in RNA samples from six neuroblastoma cell lines and of 194 genes in two commercially available reference RNA samples before and after pre-amplification, and subsequently applied on a large panel of 738 RNA samples extracted from neuroblastoma tumours. All RNA samples were evaluated for RNA integrity and purity. Starting from 5 to 50 nanograms of total RNA the sample pre-amplification method was applied, generating approximately 5 microgams of cDNA, sufficient to measure more than 1000 target genes. The results obtained from this study show a 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; no co-amplification of contaminating genomic DNA; no necessity to purify the pre-amplified material; and finally the importance of good RNA quality to enable pre-amplification.

Conclusion

Application of this unbiased and easy to use sample pre-amplification technology offers great advantage to generate sufficient material for diagnostic and prognostic work-up and enables large-scale qPCR gene-expression studies using limited amounts of sample material.