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

Objective 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. Methods 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. Results 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. Conclusions 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.

Exploring the Complexity of the Interaction between T. rubrum and S. aureus/S. epidermidis in the Formation of Polymicrobial Biofilms

“…Dermatophyte biofilm formation involves a well-coordinated process with distinct stages. Among these, adhesion is a prerequisite for biofilm development and plays a critical role in initiating the colonization of host tissues and the subsequent establishment of infection [ 50 , 51 , 52 , 53 ]. During this period, contact between the conidia, hyphal fragments, and substrate is established, accompanied by the secretion of adhesins by the arthroconidia, facilitating attachment to the host stratum corneum [ 54 ].…”

“…The temporal changes observed in this study, specifically in the population dynamics within biofilms, offer valuable insights for clinical decision-making during treatment. Prior studies have indicated that in the biofilm environment, microorganisms can secrete bioactive compounds with the potential to induce greater resistance or alterations in virulence [ 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ]. Furthermore, bridging the gap between laboratory studies and practical applications is essential for translating scientific insights into effective clinical practices.…”

Evaluation of the antidermatophytic activity of potassium salts of N-acylhydrazinecarbodithioates and their aminotriazole-thione derivatives

“…Melting curve analysis was performed by heating the amplicon from 72 to 95 °C. Relative gene expression levels were calculated according to the 2 −ΔΔCT method, with sdha and rpl 2 for T. rubrum (Supplementary Table S5 ) as the reference genes according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines 91 . Statistical analysis was performed using one-way ANOVA test using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, California, USA).…”

Effect of PLC-β1/CaM signaling pathway mediated by AT1R on the occurrence and development of hepatocellular carcinoma

“…The formed RT-PCR product was electrophoresed in a 2% agarose gel, stained with ethidium bromide (#E8751; Sigma-Aldrich; St. Louis, Mo, USA), and then photographed by a Bio-Rad gel imaging system. For Real-time PCR, 2 −ΔΔCT method was selected for real-time fluorescence quantitative detection, and then a standard curve was drawn and the relative expression of the target gene was analyzed [ 24 ].…”