GAPDH, YWHAZ, and RRN18S as control reference genes for gene expression studies on renal cell carcinoma (RCC) formaldehyde-fixed paraffin-embedded (FFPE) tissue samples.

Abstract

389 Background: It is mandatory to have a control reference gen (RG) to correctly measure gene expression by real-time quantitative PCR (qPCR). The purpose of this study was to test a panel of 12 RGs (Human Endogenous Control Gene Panel, tataabiocenter) in order to select and validate the most appropriate control genes for expression studies on FFPE RCC tissues. Methods: qPCR followed by Normfinder and geNorm-based analysis was employed (GenEx Standard). The study was performed on 9 selected RCC tumor samples with different local stage (T1, T2 and T3). The most representative RCC histologies were also collected; clear-cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC) and cromophobe renal cell carcinoma (cRCC). A commercial pool (Biochain) of 5 cases of normal kidney was analyzed too. All samples were measured in triplicate. Expression levels of RGs: GAPDH, TUBB, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP, GUSB and HPRT1 were measured by qPCR on a Light Cycler 480 (Roche) utilizing Light Cycler 480 SYBR Green I Master (Roche). Results: The analysis of experimental data showed that RRN18S is the most stable gene found in our FFEP RCC samples followed by GUSB and TBP. In contrast, ACTB was found to be the least stable gene between our samples. GAPDH together with YWHAZ was showed as the pair of genes introducing the least systematic error into data normalization (M-value < 1) needed to conduct expression gene studies further. Conclusions: These data suggest that GAPDH, YWHAZ and RRN18S are the most suitable RGs for gene expression profile studies in FFEP RCC. Standardization of RGs will help us to compare results from translational studies on RCC FFPE samples genes profiling. No significant financial relationships to disclose.