Identification of appropriate housekeeping genes for gene expression studies in human renal cell carcinoma under hypoxic conditions

Abstract

Background: Due to the loss of von Hippel-Lindau tumor suppressor function, clear renal cell carcinoma (ccRCC) deregulates hypoxia pathways. Quantitative PCR is a powerful tool for quantifying differential expression between normal and cancer cells. Reliable gene expression analysis requires the use of genes encoding housekeeping genes. Therefore, in this study, eight reference candidate genes were evaluated to determine their stability in 786-0 cells under normoxic and hypoxic conditions. Methods and Results: Four different tools were used to rank the most stable genes: GeNorm, NormFinder, BestKeeper, and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg. According to the four algorithms, the TFRC reference gene was identified as the most stable, and therefore, no agreement was observed for the 2nd and 3rd positions. A general classification was then established using the RankAggreg tool. Finally, the three most suitable reference genes to be used in 786-0 cells under normoxic and hypoxic conditions were TFRC, RPLP0, and SDHA. Conclusions: To our knowledge, this is the first study to evaluate reliable genes that can be used in gene expression analysis in ccRcc under a hypoxic environment.