Abstract
Candida tropicalis arises as one of the predominant non-Candida albicans Candida (NCAC) species causing invasive candidiasis in Asian countries. A rise in reports of C. tropicalis with a parallel increase in fluconazole resistance has also been observed. The genes and underlying pathways associated with azole antifungal resistance in C. tropicalis is still not properly understood. The RT-qPCR is the most promising approach for expression analysis of target genes to understand the mechanisms of resistance. The reliability and reproducibility of this technique depend on the selection of suitable reference genes for the normalization in expression study. The present study investigated the expression stability levels of ten genes including ACT1, EF1, GAPDH, PGK1, RDN5.8, RDN18, RDN28, SDHA, TUB1, and UBC13 for their suitability in fluconazole treated/untreated C. tropicalis. The stability levels of these genes were examined by the ∆∆CT, ΔCT, Pfaffl methods and five independent software including hkgFinder, geNorm, NormFinder, BestKeeper, and RefFinder software. We report, the EF1 and ACT1 were the most stable reference genes for normalization and can be used for the gene expression analysis in C. tropicalis. To the best of our knowledge, our study is the first to select and validate the reference genes in C. tropicalis for RT-qPCR based expression analysis.
Highlights
Candida tropicalis, a non-Candida albicans Candida (NCAC) resides in human skin, genitourinary, respiratory, and gastrointestinal tracts as a part of the normal microbiota[1,2,3]
The most frequently utilized reference genes for expression analysis, like 18S and 28S ribosomal RNAs, β-actin, tubulin, and glyceraldehyde 3-phosphate dehydrogenase, have presented variable levels of expression under different conditions in diverse cells and tissues, and are inappropriate for the normalization of RT-qPCR10,17,22–26. This suggests the necessity to select and validate the appropriate reference genes which are specific for the type of sample and experimental condition used in different studies
We examined the stability of these 10 reference genes by utilizing eight different approaches including, ∆∆CT27, ΔCT28, Pfaffl[29] methods and by using 5 different software like hkgFinder[17], geNorm[21], NormFinder[30], BestKeeper[31], and RefFinder[32]
Summary
A non-Candida albicans Candida (NCAC) resides in human skin, genitourinary, respiratory, and gastrointestinal tracts as a part of the normal microbiota[1,2,3]. The most frequently utilized reference genes for expression analysis, like 18S and 28S ribosomal RNAs, β-actin, tubulin, and glyceraldehyde 3-phosphate dehydrogenase, have presented variable levels of expression under different conditions in diverse cells and tissues, and are inappropriate for the normalization of RT-qPCR10,17,22–26. This suggests the necessity to select and validate the appropriate reference genes which are specific for the type of sample and experimental condition used in different studies. The selected stable reference genes were validated by analyzing the relative expression levels of different pleiotropic azole resistance genes by using the comparative ∆∆CT method
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