Selection of reference genes for use in quantitative reverse transcription PCR assays when using interferons in U87MG

Relative gene quantification by quantitative reverse transcription PCR (qRT-PCR) is an accurate technique only when a correct normalization strategy is carried out. Some of the most commonly genes used as reference have demonstrated variation after interferon (IFN) treatments. In this work we evaluated the suitability of seven reference genes (RGs) [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), β-2Microglobulin (B2M), ribosomal RNA subunits 18S and 28S, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) and the RNA helicase (DDX5)] for use in qRT-PCR assays in the glioblastoma-derived cell line U87MG treated with IFNα, IFNγ or a co-formulated combination of both IFNs (HeberPAG); untreated cell lines were included as control. Data was analyzed using geNorm and NormFinder softwares. The expression stability of the seven RGs decreased in order of DDX5/GAPDH/HMBS, 18S rRNA, YWHAZ, 28S rRNA and B2M. qRT-PCR analyses demonstrated that DDX5, GAPDH and HMBS were among the best stably expressed markers under all conditions. Both, geNorm and NormFinder, analyses proposed same RGs as the least variables. Evaluation of the expression levels of two target genes utilizing different endogenous controls, using REST-MCS software, revealed that the normalization method applied might introduce errors in the estimation of relative quantities. We concluded that when qRT-PCR is designed for studies of gene expression in U87MG cell lines treated with IFNs type I and II or their combinations, the use of all three GAPDH, HMBS and DDX5 (or their combinations in pairs) as RGs for data normalizations is recommended.