Evaluation of internal reference genes for photodynamic inactivation-based quantitative PCR studies in Staphylococcus aureus

<h2>Abstract</h2> <i>Staphylococcus aureus</i> is one of the most important bacterial pathogens causing severe nosocomial infections. In the laboratory, the standard and methicillin-resistant <i>S. aureus</i> (MRSA) strains can be cultivated as free-floating planktonic cells or as biofilm, often used in photodynamic inactivation (PDI) experiments, which may utilize gene expression as a major read-out. To deem expression data accurate, reference genes to be used as normalizers of quantitative real-time PCR (qPCR) data have to be stably expressed. Therefore, the goal of this study was to identify the most stably expressed reference genes in standard and MRSA strains cultivated as planktonic cells or biofilm with or without polyurethane material, modified with a photosensitizer phloxine B and subjected to PDI. Raw qPCR data of six candidate reference genes (<i>femA, glyA, hu, rho, rpoD, rrsC</i>) were analyzed with four standard algorithms, geNorm and NormFinder included in a GenEx software package, as well as with BestKeeper and ΔCq to determine the stability of the genes. Generally, the analyses' outputs showed mostly differences, which were notable between GenEx and BestKeeper. Overall, in the comprehensive gene stability analysis averaging all the analyses, the most stable genes in CCM/L18 planktonic cells were <i>rho</i>/<i>rpoD</i> (or <i>rpoD</i>/<i>femA</i> as the second best), in CCM/L18 biofilm it was <i>rho</i> (or <i>glyA</i> as a second best), and in CCM/L18 biofilm on PU the most suitable genes were <i>rpoD</i>/<i>femA</i> (or <i>glyA</i>/<i>rpoD</i> as the second best). Although the NormFinder analysis of gene expression variation, directly comparing the strains under individual experimental conditions, provided a slightly different ranking for a given strain and an experimental condition, the most stable genes match those in the comprehensive analysis as their stability values varied by a very narrow margin. In the ever-evolving field of antimicrobial research, this study contributes to the stability data on reference genes of <i>S. aureus</i> strains subjected to photosensitizer-coupled PDI treatment and underscores the necessity to validate a set of housekeeping genes before experiments.