Selection and validation of reference genes for qPCR in the human dermatophyte Trichophyton rubrum exposed to different carbon sources which promote adhesion-inducing conditions.

Abstract

The present study aimed to identify reference genes for qPCR analysis of T.rubrum growth in culture media which promote adhesion-inducing conditions to the host tissue. We investigated the suitability of six candidate reference genes: β-act, β-tub, ef1-α, gapdh, sdha and rpl2 in reference strain of Trichophyton rubrum in response to different environmental stimuli. The stability of these genes was determined by NormFinder, geNorm and BestKeeper software. Our data obtained from the three algorithms revealed that mRNA expression levels of two candidate reference genes, ef1-α and β-tub, remained the most stable in response to different carbon sources, while different sample sets had their own most stable reference genes, highlighting the importance of the choice of internal controls in qPCR experiments. We then checked the stability of ef1-α and β-tub reference genes expression in different T.rubrum strains, suggesting that these two genes are reliable for normalisation of qPCR. Finally, we validated the suitability of selected reference genes as internal controls for target gene (SUB3) using the 2-ΔΔCt method. The best result indicating an increase of SUB3 transcript of T.rubrum was found when the two the most stable reference (ef1-α and β-tub ) genes were used, as revealed by all three algorithms. We recommend the use of ef1-α and β-tub as reference genes for qPCR analysis of target gene expression in T.rubrum exposed to different carbon sources which promote adhesion-inducing conditions.