Abstract
Hypoxia pathways are deregulated in clear renal cell carcinoma (ccRCC) because of the loss of the von Hippel-Lindau tumor suppressor function. 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. 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 RankAggreg. According to the four algorithms, the TFRC reference gene was identified as the most stable. There was no agreement among the results from the algorithms for the 2nd and 3rd positions. A general classification was then established using the RankAggreg tool. Finally, the three most suitable reference genes for use in 786-0 cells under normoxic and hypoxic conditions were TFRC, RPLP0, and SDHA. To the best of our knowledge, this is the first study to identify reliable genes that can be used for gene expression analysis in ccRCC in a hypoxic environment.
Highlights
Renal cell carcinoma (RCC) is a group of malignant histological subtypes that arise from epithelial cells, accounting for 2%–3% of all malignancies in adults [1,2]
The three most suitable reference genes to be used in 786-0 cells under normoxic and hypoxic conditions were TFRC, RPLP0, and SDHA
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
Summary
Renal cell carcinoma (RCC) is a group of malignant histological subtypes that arise from epithelial cells, accounting for 2%–3% of all malignancies in adults [1,2]. The majority of ccRCC patients have a mutation in the von Hippel-Lindau (VHL) gene, which is located on the short arm of chromosome 3 and serves as an autosomal dominant tumor suppressor [5]. HIF is a heterodimeric transcription factor (HIF1α and HIF1β) that coordinates the expression of several genes responsible for cellular adaptation to hypoxia [6]. HIF1α protein is hydroxylated, recognized by pVHL, which drives them to degradation. In RCC patients, pVHL is nonfunctional and unable to target HIF1α protein for degradation. Due to the loss of von Hippel-Lindau tumor suppressor function, clear renal cell carcinoma (ccRCC) deregulates hypoxia pathways. In this study, eight reference candidate genes were evaluated to determine their stability in 7860 cells under normoxic and hypoxic conditions
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