phaidra.vetmeduni.ac.at o:3998 DOI 10.3390/toxins17020058 Establishing Reference Genes for Accurate Gene Expression Profiling in Toxigenic Bacillus cereus 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. eng 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 2025-04-02T08:47:20.065Z LOMv1.0 Author BEGIN:VCARD VERSION:3.0 N:Edelbacher;Tanja V.; FN:Tanja V. Edelbacher X-ORCID:https://orcid.org/0000-0002-0009-0004 END:VCARD BEGIN:VCARD VERSION:3.0 N:Laimer-Digruber;Astrid; FN:Astrid Laimer-Digruber X-ORCID:https://orcid.org/0000-0002-8809-2530 END:VCARD BEGIN:VCARD VERSION:3.0 N:Pfaffl;Michael W.; FN:Michael W. Pfaffl X-ORCID:https://orcid.org/0000-0002-3192-1019 END:VCARD BEGIN:VCARD VERSION:3.0 N:Ehling-Schulz;SandraMonika; FN:SandraMonika Ehling-Schulz X-ORCID:https://orcid.org/0000-0001-7384-0594 END:VCARD https://w3id.org/kim/hcrt/scheme https://w3id.org/kim/hcrt/text Textdokument Text LOMv1.0 yes https://creativecommons.org/licenses/by/4.0 application/pdf 1478841 https://phaidra.vetmeduni.ac.at/o:3998 https://phaidra.vetmeduni.ac.at/api/object/o:3998/thumbnail LOMv1.0 discipline