Selection and Validation of Reference Genes for qPCR Analysis Under 409 nm and 415 nm Antimicrobial Blue Light Exposure

Antimicrobial blue light (aBL) is a promising alternative to combat multidrug-resistant bacteria. Stable reference genes are essential for accurate gene expression analysis under stress conditions such as aBL exposure. This study addresses the lack of information on appropriate reference genes for aBL-treated Escherichia coli. Candidate genes were analyzed for stability under 409 and 415 nm aBL using BestKeeper, geNorm, NormFinder, and RefFinder algorithms. The gene encoding the integration host factor β subunit (ihfB) was identified as the most stable reference gene, followed by cysG, uidA, and gyrA. Minimal differences in gene expression were observed between wavelengths, with a significant increase in oxyR (oxidative stress regulator) at 409 nm. ihfB is recommended for reliable normalization in aBL gene expression studies, enhancing the accuracy of transcriptomic analyses in this emerging antimicrobial strategy.