title: Measurable impact of RNA quality on gene expression results from quantitative PCR.
abstract: 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 of 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 normalization factor based on the mean expression level of four 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 reverse transcription quantitative PCR based results in relation to RNA quality.
authors.affiliation: Centre for Medical Genetics, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium.
authors.forename: Joëlle
authors.initials: J
authors.lastname: Vermeulen
authors.forename: Katleen
authors.initials: K
authors.lastname: De Preter
authors.forename: Steve
authors.initials: S
authors.lastname: Lefever
authors.forename: Justine
authors.initials: J
authors.lastname: Nuytens
authors.forename: Fanny
authors.initials: F
authors.lastname: De Vloed
authors.forename: Stefaan
authors.initials: S
authors.lastname: Derveaux
authors.forename: Jan
authors.initials: J
authors.lastname: Hellemans
authors.forename: Frank
authors.initials: F
authors.lastname: Speleman
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D045744:Cell Line, Tumor; D046209:Electrophoresis, Microchip; D020869:Gene Expression Profiling / Q000592:standards*; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis / Q000592:standards*; D012334:RNA, Neoplasm / Q000032:analysis / Q000592:standards; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D014408:Biomarkers, Tumor; D012333:RNA, Messenger; D012334:RNA, Neoplasm