Selection and validation of reference genes for qRT-PCR analysis of gene expression in Microsporum canis growing under different adhesion-inducing conditions

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.

Figure 1

Expression level of nine reference genes in M. canis. The mean C_t values for all experimental conditions of each candidate reference gene are shown as box plot representations. The box indicates the 25^th and 75^th percentiles, the line across the box represents the median and whisker caps the maximum and minimum values.

Figure 2

Melting curves of the nine M. canis candidate reference genes show single peaks (A). 8% polyacrylamide gel electrophoresis indicated the amplification of a single product of the expected size for nine reference genes (B).

Figure 3

Gene expression stability (M-value) and pairwise variation (V_n/V_n+1) of the Microsporum canis candidate reference genes analyzed by geNorm.

Figure 4

Stability of mbp1, β-act and sdha reference genes expression, evaluated in three M. canis strains cultured on MM-Cove medium (control medium) and MM-Cove medium supplemented with keratin. Gene expression levels are represented by average C_t values. mbp1 (p = 0.91, ANOVA), β-act (p = 0.46, ANOVA) and sdha (p = 0.93, ANOVA) gene expression level was not significantly different across analyzed culture conditions. Error bars indicate standard error.

Figure 5

Relative quantification of MEP3 gene expression in control, keratin and keratin/soy protein samples using different reference gene variants: A (p = 0.67, ANOVA) - two least stable reference genes rpl2 and rGTP; B (p = 0.72, ANOVA) – three most stable reference genes mbp1, β-act and sdha; C (p = 0.55, ANOVA) – all candidate reference genes. Error bars indicate standard error.