Normalizing to GADPH jeopardises correct quantification of gene expression in ovarian tumours – IPO8 and RPL4 are reliable reference genes

BACKGROUND To ensure a correct interpretation of results obtained with quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR), it is critical to normalize to a reference gene with stable mRNA expression in the tissue of interest. GADPH is widely used as a reference gene in ovarian tumour studies, although lacking tissue-specific stability. The aim of this study was to identify alternative suitable reference genes for RT-qPCR studies on benign, borderline, and malignant ovarian tumours. METHODS We assayed mRNA levels for 13 potential reference genes - ABL1, ACTB, CDKN1A, GADPH, GUSB, HPRT1, HSP90AB, IPO8, PPIA, RPL30, RPL4, RPLPO, and TBP -with RT-qPCR in 42 primary ovarian tumours, using commercially pre-designed RT-qPCR probes. Expression stability was subsequently analysed with four different statistical programs (GeNorm, NormFinder, BestKeeper, and the Equivalence test). RESULTS Expression of IPO8, RPL4, TBP, RPLPO, and ACTB had the least variation in expression across the tumour samples according to GeNorm, NormFinder, and BestKeeper. The Equivalence test found variation in expression within a 3-fold expression change between tumour groups for: IPO8, RPL40, RPL30, GUSB, TBP, RPLPO, ACTB, ABL1, and CDKN1A. However, only IPO8 satisfied at a 2-fold change as a cut-off. Overall, IPO8 and RPL4 had the highest, whereas GADPH and HPRT1 the lowest expression stability. Employment of suitable reference genes (IPO8, RPL4) in comparison with unsuitable ones (GADPH, HPRT1), demonstrated divergent influence on the mRNA expression pattern of our target genes - GPER and uPAR. CONCLUSIONS We found IPO8 and RPL4 to be suitable reference genes for normalization of target gene expression in benign, borderline, and malignant ovarian tumours. Moreover, IPO8 can be recommended as a single reference gene. Neither GADPH nor HPRT1 should be used as reference genes in studies on ovarian tumour tissue.