Abstract
Tinea pedis is a type of dermatophytosis caused by anthropophilic keratinolytic fungi such as Trichophyton interdigitale. Quantitative reverse transcription PCR (RT-qPCR) is a reliable and reproducible technique for measuring changes in target gene expression across various biological conditions. A crucial aspect of accurate normalization is the choice of appropriate internal controls. To identify reference genes for accurate evaluation of expression levels in T. interdigitale, the transcription levels of eight candidate reference genes (adp-rf, β-act, ef1-α, gapdh, psm1, sdha, rpl2 and ubc) and one target gene (Tri m4) were analysed by RT-qPCR after growing the dermatophyte under different environmental conditions. The results obtained from expression stability evaluations with NormFinder, geNorm, BestKeeper, and RefFinder software demonstrated that adp-rf and psm1 were the most stable internal control genes across all experimental conditions. The present study constitutes the first report of the identification and validation of reference genes for RT-qPCR normalization for T. interdigitale grown under different environmental conditions resembling the conditions encountered by fungi during invasion of skin.
Highlights
Trichophyton interdigitale is a keratinophilic and keratinolytic fungus belonging to the dermatophyte group[1], and it is responsible for infections of the feet and toes[2]
The GeNorm algorithm, which is a module of qbase + (Biogazelle), was used to evaluate the candidate reference genes based on their expression stability values (M-values) and pairwise variations (Vn/Vn+1)
The pairwise variation (Vn/Vn+1) results indicated that five reference genes should be used for reliable normalization (V5/6 = 0.136) (Fig. 3)
Summary
Trichophyton interdigitale is a keratinophilic and keratinolytic fungus belonging to the dermatophyte group[1], and it is responsible for infections of the feet and toes (tinea pedis)[2]. Dermatophytes have been recorded worldwide with variations in epidemiology, distribution, incidence and target hosts from one location to another Different conditions, such as geographic location, climate, health care quality, immigration status, hygiene culture, and socioeconomic status, may influence the development of dermatophyte infections[7]. Due to the limited knowledge regarding reference genes useful for RT-qPCR analysis in dermatophytes[10] and the insufficient information on the complete genome sequence of T. interdigitale, eight reference genes, including adp-rf (ADP ribosylation factor), β-act (β-actin), ef1-α (elongation factor 1-alpha), gapdh (glyceraldehyde 3-phosphate dehydrogenase), psm[1] (mitotic cohesion complex subunit Psm1), sdha (succinate dehydrogenase complex flavoprotein subunit A), rpl[2] (ribosomal protein L2) and ubc (ubiquitin) (Table 1) were selected and evaluated in a T. interdigitale strain subjected to 13 different environmental conditions (Table 2). The online comprehensive tool RefFinder was used to compare and rank the candidate reference genes
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