Reference genes for accurate evaluation of expression levels in Trichophyton interdigitale grown under different carbon sources, pH levels and phosphate levels

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.

incorrect data analysis. Therefore, it is necessary to establish a set of optimal reference genes before conducting target gene expression analysis. Due to the limited knowledge regarding reference genes useful for RT-qPCR analysis in dermatophytes 10 and the particularly 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), psm1 (mitotic cohesion complex subunit Psm1), sdha (succinate dehydrogenase complex flavoprotein subunit A), rpl2 (ribosomal protein L2) and ubc (ubiquitin) ( Table 1) were ultimately selected and evaluated in a T. interdigitale strain subjected to 13 different environmental conditions ( Table 2). The selected candidate reference genes were chosen from among internal controls used in some species of fungi, including dermatophytes 10,11 , and in other eukaryotic organisms 9,10,12-15 . The expression stability of each candidate reference gene was calculated by the following algorithms: the geNorm module of qbase + (Biogazelle) 16 , NormFinder 17 , and BestKeeper 18 . The online comprehensive tool RefFinder was ultimately used to compare and rank the candidate reference genes.
According to NormFinder 17 , across all experimental conditions, psm1 had the lowest stability value (SV = 0.080) (Fig. 2). Psm1 and rpl2 constituted the best combination of internal control genes with SV = 0.061 under all experimental conditions. Psm1 was found to be the most stably expressed gene in the presence of colloidal chitin (SV = 0.032) ( Assessment of the expression variation of the candidate reference genes using the BestKeeper algorithm 18 revealed that seven of the genes had standard deviation values defined as acceptable [0.5 < SD[±C t ] ≤ 1.00], while gapdh had an unacceptable standard deviation, as it was higher than 1.0 (SD = 1.32) ( Table 3). Analyses showed also significant expression correlations with the BestKeeper index, which is the geometric mean of the C t values of the analysed genes (correlation coefficient r = 0.698-0.901), except for the β-act gene (r = 0.145). The expression of all genes correlated with the BI with p values < 0.001, except the β-act gene (p-value = 0.543). The overall order of the most stable genes based on BestKeeper was psm1, adp-rf, ef1-α, sdha, ubc and rpl2 (Fig. 2, Table 4).
In the final step, RefFinder, a free online tool for the identification of stable reference genes that integrates all methods applied in the present study, was used to generate a final ranking of the eight reference genes according to their geomean ranking values. As shown in Fig. 2, psm1 and adp-rf were ranked as the best reference genes for measuring target gene expression levels under the chosen conditions. Stability and validation of adp-rf and psm1 as reference genes. To confirm adp-rf and psm1 as the most stable reference genes, their expression was compared in the T. interdigitale CBS 124408 reference strain and two clinical isolates: T. interdigitale 12/2010 and T. interdigitale 45/10. These three strains of T. interdigitale were incubated at 28 °C for 48 h in control medium and in medium supplemented with keratin. The obtained C t values (Fig. 5) for the adp-rf and psm1 genes were not significantly different under both conditions (p psm1 = 0.93; p adp-rf = 0.89, ANOVA) (Fig. 5), which confirmed that these reference genes can be used for accurate expression level evaluation in various T. interdigitale strains. To confirm the reliability of adp-rf and psm1 as reference genes for RT-qPCR normalization, the expression of Tri m4 was examined 19 . Tri m4 is known as an aminopeptidase gene whose expression increases in the presence of keratin and elastin, which suggests that the product of this gene may play an important role as a virulence factor 19 . The validation was performed using templates from the T. interdigitale 45/10 strain incubated at 28 °C for 48 h in control medium (MM-Cove) and in medium supplemented with keratin or elastin. Three different sets of reference genes were analysed: set A included the most stable reference genes (adp-rf and psm1), set B included the least stable reference genes (β-act and gapdh), and set C included all eight candidate reference genes. The relative expression of the target gene was determined using the 2 −ΔΔCt method 20 . As shown in Fig. 6, an increase in Tri m4 transcript levels in T. interdigitale growing in the presence of keratin or elastin in relation to control conditions was noticed only when the adp-rf and psm1 reference genes (set A) previously selected by all four algorithms were used.

Discussion
Quantitative reverse transcription PCR (RT-qPCR) is an efficient method for analysing target gene expression but requires comprehensive normalization with properly selected reference genes. The internal controls should have relatively stable expression levels in different types of cells or tissues, and their expression should be constant under various growth conditions 21 . Many reports on target gene expression analysis have used a single housekeeping gene for RT-qPCR normalization, such as 18S rRNA, gapdh, β-act, β-tub or ef1-α 22 ; these genes www.nature.com/scientificreports www.nature.com/scientificreports/ regulate basic cellular functions. Such an approach has a strong tradition and history of use since the introduction of reverse-transcription-based assays. However, since 2009, according to the MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments 21 , no single reference gene should be used to quantify target gene expression under different conditions, and candidates for reference genes should be carefully selected for each study to comply with these guidelines. Numerous studies have shown that the expression of the above housekeeping genes can vary between individual tissues or experimental conditions such that no universal reference gene can be used in all situations 18,[23][24][25][26][27] . Moreover, a reference gene with stable expression in one organism may not be suitable for normalization of gene expression in another organism, even a closely  Table 3. Gene expression stability (M-value) of each Trichophyton interdigitale candidate reference gene as analysed by geNorm for each experimental condition. www.nature.com/scientificreports www.nature.com/scientificreports/ related organism. Additionally, genes such as 18S rRNA and 28S rRNA, despite their stability, are often expressed at very high levels and thus should not be used as internal controls 28,29 . To avoid biased normalization, following the MIQE guidelines 21 , the use of multiple candidate reference genes is highly recommended to obtain reliable RT-qPCR results. Based on the limited literature reports regarding the analysis of gene expression in dermatophytes 10,11 , supplemented with information on reference genes used in studies on other eukaryotes 9,12,13 , 12 genes, including adp-rf (ADP ribosylation factor), β-act (β-actin), β-tub (β-tubulin), ef1-α (elongation factor 1-alpha), gapdh (glyceraldehyde 3-phosphate dehydrogenase), mbp1 (multiubiquitin chain-binding protein 1),  Table 4. Descriptive statistics for the candidate reference genes calculated using BestKeeper. n, number of samples (three biological replicates and 30 different conditions); GM (C t ), geometric mean of C t ; AM (C t ), arithmetic mean of C t ; Min (C t ) and Max (C t ), extreme values of C t ; SD (±C t ), standard deviation of C t ; CV (% C t ), coefficient of variation expressed as a percentage of the C t value; Min (fold change) and Max (fold change), extreme value of the expression level expressed as the absolute fold change of down-or upregulation; SD (±fold change), standard deviation of the absolute fold change; BI Index (r), correlation between BestKeeper index and the contributing gene.  www.nature.com/scientificreports www.nature.com/scientificreports/ fis1 (mitochondrial fission 1 protein), psm1 (mitotic cohesion complex subunit Psm1), rGTPa (rho GTPase activating-protein 5), rpl2 (ribosomal protein L2), sdha (succinate dehydrogenase complex flavoprotein subunit A) and ubc (ubiquitin) ( Table 1 and Supplementary Table S2), were selected as putative candidates based on a BLAST search of available T. interdigitale genomic sequences. Despite several attempts at primer modification, only 8 candidate amplification products were obtained, and these genes were used in further studies. Unfortunately, only three genomes of this species are currently available in the databases, of which two are at a scaffold level, while the third is at a contig level. An in-depth analysis of these genomes revealed the presence of regions of predicted sequences, indicating that the full nucleotide sequences have yet to be established (https:// www.ncbi.nlm.nih.gov/genome/genomes/44693). It can therefore be assumed that both these problems and others related to the difficulties in correctly determining the taxonomic affiliation of many strains of T. interdigitale, as described in the literature 30 (which may affect the correct genome assembly of this species), were responsible for the unsuccessful attempts to develop a larger number of correct primers, making it impossible to test a greater number of putative reference genes.
In this study, the geNorm, NormFinder, BestKeeper and RefFinder algorithms were used to evaluate the selected candidate reference genes as internal controls for analysis of target gene expression in T. interdigitale growing under different environmental stimuli, such as supplementation with various carbon sources, low Pi, and different pH values 10 . Some of these circumstances have been suggested to promote adhesion to the host tissue and are essential for the expression of specific genes associated with adaptation and interactions between T. interdigitale and its host. Our study is the first report on the identification and validation of reference genes for T. interdigitale that indicates psm1 and adp-rf as the most stable genes among the analysed candidates (Fig. 2).
Mitotic cohesion complex ATPase subunit (psm1) is involved in mitotic cohesion loading/unloading and is required for the cohesion of sister chromatids after DNA replication. In addition, ADP-ribosylation factor (adp-rf) is a ubiquitous GTP-binding protein essential for mitotic growth. These two candidates were found in the present study to be reliable internal controls for accurate expression level analysis of target genes of T. interdigitale growing under adhesion-inducing conditions.
Llanos et al. 31 showed that according to geNorm analysis, psm1, ubcB (ubiquitin carrier protein) and sac7 (Rho GTPase activator) were ranked as the most stably expressed reference genes in the fungus Talaromyces versatilis grown under various conditions, such as in the presence of different carbon sources; under different temperatures and pH levels; and under salt stress and carbon/nitrogen starvation. However, in our previous report on the validation of reference genes for the dermatophyte Microsporum canis 11 , psm1 was classified in the group of unstable reference genes. To date, there have been only two studies on the validation of psm1 as a stably expressed reference gene in RT-qPCR analysis.
The ADP ribosylation factor gene (adp-rf) was found to be the best reference gene for analyses of target transcript levels in an exotic invasive insect, Leptinotarsa decemlineata 32 ; the melon Cucumis melo L. 33 ; the cereal wheats Triticum spp. 12 , the Pacific oyster, Crassostrea gigas 34 ; the monkey Macaca fascicularis 35 ; and the desert willow shrub, Salix psammophila 36 . Again, our previous study 11 demonstrated that the expression stability of adp-rf was low in M. canis. Furthermore, the β-act gene, which encodes a cytoskeletal protein involved in many cellular processes, and the gapdh gene, which encodes an enzyme of the glycolytic pathway, are often used as reliable reference genes in expression analyses 37 ; however, these genes were in the group of the least stable reference genes in the present study. On the other hand, in the search for reliable reference genes for RT-qPCR analysis of target gene expression in M. canis, the β-act gene was classified as one of the three most stable genes 11 . The present and the previous results 11 of our team confirmed that the stability of housekeeping gene expression should be verified for each condition and each particular species (Table 3, Fig. 4), which again highlights the fact that there is no ideal and universal internal control gene for RT-qPCR analysis.
To validate the reference genes selected by the four algorithms, the genes were used as reference genes for the measurement of the relative expression of Tri m4, a gene that encodes aminopeptidase and is known to be upregulated in the presence of keratin and elastin as inducers 19 . Elevated Tri m4 expression was detected in T. interdigitale growing under inducing conditions only when the two internal controls psm1 and adp-rf, which were selected as the most stable internal controls by the four algorithms, were used in combination (Fig. 6). Upregulation was detected neither for the least stable pair of reference genes (set B) nor for the whole set of eight candidate genes (set C). These results clearly confirmed that the chosen best pair of internal control genes can be preferentially used for RT-qPCR normalization in the case of T. interdigitale grown under the described experimental conditions.

Conclusion
The present study was the first attempt to identify and validate T. interdigitale internal control genes. The psm1 and adp-rf genes were found to be the most stable reference genes appropriate for gene expression analysis in T. interdigitale. The use of these genes as internal controls may further improve the robustness of RT-qPCR for T. interdigitale grown under adhesion-inducing conditions.