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.

Comparative assessment of three RNA extraction methods for obtaining high-quality RNA from Candida viswanathii biomass.

Isolating high quality RNA is a limiting factor in molecular analysis, since it is the base for transcriptional studies. The RNA extraction method can directly affect the RNA quality and quantity, as well as, its overall cost. The industrial importance of the yeast genus Candida in several sectors comes from their capacity to produce Lipases. These enzymes are one of the main metabolites produced by some Candida species, and it has been shown that Candida yeast can biodegrade petroleum hydrocarbons and diesel oil from biosurfactants that they can produce, a feature that turns these organisms into potential combatants for bioremediation techniques. Thus, this study aimed to determine an efficient method for isolating high quality RNA from Candida viswanathii biomass. To achieve this aim, three different RNA extraction methods, TRIzol, Hot Acid Phenol, and CTAB (Cetyltrimethylammonium Bromide), were tested. The three tested methods allowed the isolation of high-quality RNA from C. viswanathii biomass and yielded suitable RNA quantity for carrying out RT-qPCR studies. In addition, all methods displayed high sensitivity for the expression analysis of the CvGPH1 gene through RT-qPCR, with TRIzol and CTAB showing the best results and the CTAB method displaying the best cost-benefit ratio (US$0.35/sample).