Abstract
Dermatophytes are the group of filamentous fungi infecting keratinized structures such as skin, hair, and nails. Knowledge about genes and molecular mechanisms responsible for pathogenicity, as well as other biological properties of Microsporum canis is still relatively poor. The qRT-PCR is a reliable technique for quantifying gene expression across various biological processes, and choosing a set of suitable reference genes to normalize the expression data is a crucial step of this technique. We investigated the suitability of nine candidate reference genes: β-act, β-tub, adp-rf, ef1-α, sdha, rpl2, mbp1, psm1, and rGTPa for gene expression analysis in the dermatophyte M. canis in response to different carbon sources, phosphate levels, and pH shifts - factors that are extremely important and necessary for growth of dermatophyte in the host tissue. The transcription stability of these genes was evaluated using NormFinder, geNorm, BestKeeper, and RefFinder software. Regarding expression stability, mbp1, β-act, and sdha were the most stable housekeeping genes which we recommend for future qRT-PCR studies on M. canis strains. To the best of our knowledge this is the first study on selection and validation of reference genes for qRT-PCR data normalization in M. canis growth in culture media which promote adhesion-inducing conditions.
Highlights
Effective means of studying response to different environmental stimuli and a necessary component in identifying genes and regulatory mechanisms associated with biological processes in any organism
The candidate reference genes exhibited Ct values ranging from 15.08–26.65 (Fig. 1, Table 1)
The expression stability of candidate reference genes needs to be verified before each qRT-PCR experiment
Summary
Effective means of studying response to different environmental stimuli and a necessary component in identifying genes and regulatory mechanisms associated with biological processes in any organism. Due to the limited knowledge on such genes suitable for expression analysis in dermatophytes[24] we investigated the transcription level of a group of nine candidate housekeeping genes: β-act (β-actin), β-tub (β-tubulin), adp-rf (ADP ribosylation factor), ef1-α (elongation factor 1-alpha), sdha (succinate dehydrogenase complex flavoprotein subunit A), rpl[2] (ribosomal protein L2), mbp[1] (multiubiquitin chain binding protein 1), psm[1] (mitotic cohesion complex subunit Psm1), rGTPa (rho GTPase activating-protein 5) (Table 1) as reference genes for selected clinical strain of M. canis grown under different adhesion-inducing environmental stimuli typical for the stage of host infection, such as: different carbon sources (glucose, keratin, keratin with soy protein, elastin, collagen, colloidal chitin, keratinocyte free medium), low-Pi environments, pH shifts. The candidate reference genes were selected from among endogenous controls in some species of fungi as well as in other organisms[22,24] and to the best of our knowledge this is the first such complex search in case of dermatophyte species. The stability of each candidate reference gene was evaluated by algorithms: geNorm module of qbase + (Biogazelle), NormFinder, BestKeeper, and RefFinder web-based comprehensive tool
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