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

Matheus Martins Daúde; Ronan Cristhian Teixeira; Carlos Henrique Cardon; Gessi Carvalho de Araujo Santos; Alex Fernando de Almeida; Antonio Chalfun-Junior; Horllys Gomes Barreto

doi:10.1016/j.mimet.2023.106777

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

J Microbiol Methods. 2023 Aug:211:106777. doi: 10.1016/j.mimet.2023.106777. Epub 2023 Jul 5.

Authors

Matheus Martins Daúde¹, Ronan Cristhian Teixeira², Carlos Henrique Cardon³, Gessi Carvalho de Araujo Santos¹, Alex Fernando de Almeida², Antonio Chalfun-Junior³, Horllys Gomes Barreto⁴

Affiliations

¹ Laboratory of Molecular Analyses, Department of Life Sciences, Faculty of Medicine, Federal University of Tocantins, University Campus of Palmas, Palmas, TO, Brazil.
² Laboratory of Biotechnology, Food analyses, and Product Purification, Federal University of Tocantins, University Campus of Gurupi, TO, Brazil.
³ Laboratory of Plant Molecular Physiology, Department of Biology, Federal University of Lavras, MG, Brazil.
⁴ Laboratory of Molecular Analyses, Department of Life Sciences, Faculty of Medicine, Federal University of Tocantins, University Campus of Palmas, Palmas, TO, Brazil. Electronic address: horllys@uft.edu.br.

PMID: 37419333
DOI: 10.1016/j.mimet.2023.106777

Abstract

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.

Keywords: Carbon source; Endogenous gene; Housekeeping genes; Lipase; Normalization; Real-time PCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Gene Expression
Gene Expression Profiling*
Olive Oil
Real-Time Polymerase Chain Reaction / methods
Reference Standards
Reproducibility of Results
Triolein*

Substances

Olive Oil
Triolein

Supplementary concepts

Candida viswanathii