https://phaidra.vetmeduni.ac.at/o:3998 Establishing Reference Genes for Accurate Gene Expression Profiling in Toxigenic Bacillus cereus Edelbacher, Tanja V. Tanja V. Edelbacher 0000-0002-0009-0004 University of Veterinary Medicine Vienna Laimer-Digruber, Astrid Astrid Laimer-Digruber 0000-0002-8809-2530 University of Veterinary Medicine Vienna Pfaffl, Michael W. Michael W. Pfaffl 0000-0002-3192-1019 Ehling-Schulz, SandraMonika SandraMonika Ehling-Schulz 0000-0001-7384-0594 University of Veterinary Medicine Vienna MDPI journal article open access eng Bacillus cereus is a Gram-positive pathogen associated with foodborne illnesses and severe non-gastrointestinal infections. Robust tools for accurate gene transcription analysis are essential for studying toxin gene expression dynamics and deciphering the complex regulatory networks orchestrating the expression of toxin and virulence factors. This study aimed to identify reliable reference genes for normalizing reverse transcription quantitative PCR (RT-qPCR) data in toxigenic B. cereus. An emetic and an enteropathogenic strain were used as model organisms to establish a suitable reference gene set to monitor the dynamics of toxin gene transcription. Ten candidate reference genes were evaluated for their expression stability using geNorm, NormFinder, BestKeeper and the ?Cq method, with the final rankings integrated via RefFinder. Among the tested genes, rho, rpoD and recA were identified as the most stable expressed reference genes across all tested conditions. As shown in this proof-of-principle study, the established reference gene set provides a suitable tool to investigate the influence of extrinsic and intrinsic factors on toxin gene transcription. In conclusion, our newly established reference gene set provides a robust basis for studying toxin gene expression in B. cereus and contributes to a better understanding of its pathogenicity and potential strategies to mitigate its harmful effects. Bacillus Cereus Genetics Bacillus Cereus Metabolism Gene Expression Profiling Standards Bacterial Toxins Genetics Gene Expression Regulation, Bacterial Bacterial Proteins Genetics Reference Standards Genes, Bacterial Real-Time Polymerase Chain Reaction Standards http://creativecommons.org/licenses/by/4.0/ https://phaidra.vetmeduni.ac.at/api/object/o:3998/download 10.3390/toxins17020058 https://phaidra.vetmeduni.ac.at/o:605 application/pdf Toxins issn:2072-6651 VoR Toxins 17 2 1.41 MB 2025