Validation of endogenous control reference genes for normalising gene expression in studies of endometrial carcinoma.

Real time quantitative RT-PCR (qRT-PCR) is a powerful technique used for the relative quantification of target genes, using reference (housekeeping) genes for normalisation to ensure the generation of accurate and robust data. A systematic examination of the suitability of endogenous reference genes for gene expression studies in endometrial cancer tissues is absent. The aims of this study were therefore to identify and evaluate from the thirty-two possible reference genes from a TaqMan® array panel their suitability as an internal control gene. The mathematical software packages geNorm qBasePLUS identified Pumilio homolog 1 (Drosophila) (PUM1), ubiquitin C (UBC), phosphoglycerate kinase (PGK1), mitochondrial ribosomal protein L19 (MRPL19) and peptidylpropyl isomerase A (cyclophilin A) (PPIA) as the best reference gene combination, whilst NormFinder identified MRPL19 as the best single reference gene, with importin 8 (IPO8) and PPIA being the best combination of two reference genes. BestKeeper ranked MRPL19 as the most stably expressed gene. In addition, the study was validated by examining the relative expression of a test gene, which encodes the cannabinoid receptor 1 (CB1). A significant difference in CB1 mRNA expression between malignant and normal endometrium using MRPL19, PPIA, and IP08 in combination was observed. The use of MRPL19, IPO8 and PPIA were identified as the best reference gene combination for the normalisation of gene expression levels in endometrial carcinoma. This study demonstrates that the arbitrary selection of endogenous control reference genes for normalisation in qRT-PCR studies of endometrial carcinoma, without validation, risks producing inaccurate data and should therefore be discouraged.
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