Normalizing to GADPH jeopardises correct quantification of gene expression in ovarian tumours – IPO8 and RPL4 are reliable reference genes
BackgroundTo 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.MethodsWe 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).ResultsExpression 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.ConclusionsWe 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.
[…] Descriptive statistics, F-test for Ct variance equality and Kolmogorov-Smirnov test for normality of log-transformed relative expression values were calculated by software SPSS 19.0 (SPSS Inc, Chicago, IL). The Equivalence test [-] and statistical applets BestKeeper , geNorm , and NormFinder  were used for analysis of genes expression stability. GeNorm calculates a gene-stability measure, M-value, as the average pair-wise variation of a particular gene to all other candidate reference genes . On the other hand, the stability value calculated with NormFinder combines estimated both intra-group and inter-group variations . Genes with the lowest M-values have the most stable expression (least variability). Relative expression values for target genes were analysed by Kruskal-Wallis and Mann–Whitney tests, and the log-transformed values by one-way ANOVA. P < 0.05 was considered significant. […]