Selection and validation of reference genes for RT-qPCR gene expression studies in Candida viswanathii cultivated under different grown conditions.

The properties presented by Candida viswanathii's lipases turn this specie into a promising producer of potentially applicable lipases in several industrial sectors, such as: food, textiles, in the oleochemical and paper industries, and also in different pharmaceutical applications. However, studies for elucidating growth and developmental processes at the molecular level in this species are still incipient. Performing such kinds of studies often rely on the use of the RT-qPCR, which is a highly sensitivity technique, but whose parameters must be carefully planned for achieving reliable data. Among the crucial parameters required for achieving reliable results through this technique, the use of appropriated and validated reference genes is one the most important, constituting a bottleneck, mainly in species where molecular studies are scarce. Thus, the aim of this study was to determine the best reference genes for RT-qPCR gene expression studies in C. viswanathii grown in culture media containing four different carbon sources (Olive oil, Triolein, Tributyrin, and Glucose). Eleven candidate reference genes (ACT, GPH1, AGL9, RPB2, SAP1, PGK1, TAF10, UBC13, TFC1, UBP6, and FBA1) were analyzed for their expression patterns and stability. Analysis of gene expression stability was performed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms, and validation of the results was performed through analyzing the expression of a lipase gene, CvLIP4. Analyzing the four treatments together, CvACT and CvRPB2 constituted the best reference gene pair. When treatments are analyzed individually, CvRPB2/CvACT, CvFBA1/CvAGL9, CvPGK1/CvAGL9 and CvACT/CvRPB2 were the best reference gene pairs for the culture media containing olive oil, triolein, tributyrin, and glucose as carbon sources, respectively. These results are essential and form the basis for the development of relative gene expression studies in C. viswanathii, since adequate reference genes are crucial for the reliability of RT-qPCR data.