Measurable impact of RNA quality on gene expression results from quantitative PCR

Joëlle Vermeulen1, Katleen De Preter1, Steve Lefever1, Justine Nuytens1, Fanny De Vloed1, Stefaan Derveaux1, Jan Hellemans2, Frank Speleman1, Jo Vandesompele2
1Ghent University, Ghent, Belgium; 2Biogazelle, Zwijnaarde, Belgium & Ghent University, Ghent, Belgium

Compromised RNA quality is suggested to lead to unreliable results in gene expression studies. Therefore, assessment of RNA integrity and purity is deemed essential prior to including samples in the analytical pipeline. This may be of particular importance when diagnostic, prognostic or therapeutic conclusions depend on such analyses. In this study, the comparative value of six RNA quality parameters was determined using a large panel of 740 primary tumour samples for which real-time quantitative PCR gene expression results were available. The tested parameters comprise microfluidic capillary electrophoresis based 18S/28S rRNA ratio and RNA Quality Index value, HPRT1 5’-3’ difference in quantification cycle (Cq) and HPRT1 3’ Cq value based on a 5’/3’ ratio mRNA integrity assay, the Cq value of expressed Alu repeat sequences, and a normalisation factor based on the mean expression level of 4 reference genes. Upon establishment of an innovative analytical framework to assess impact of RNA quality, we observed a measurable impact of RNA quality on the variation of the reference genes, on the significance of differential expression of prognostic marker genes between two cancer patient risk groups, and on risk classification performance using a multigene signature. This study forms the basis for further rational assessment of RT-qPCR based results in relation to RNA quality.

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