Stability assessment of housekeeping genes for qRT-PCR in cultured at 22°C and 37°C.

, a species within the genus , thrives optimally at 22-25°C but can also grow at the mammalian core body temperature of 37°C. This dual temperature adaptability necessitates establishing both temperature conditions in research to examine the effects on various biological processes. In quantitative real-time PCR (qRT-PCR) assays, the selection of appropriate housekeeping genes is vital for data accuracy. Nevertheless, the lack of alternatives and information often leads to the default use of the 16S rRNA gene despite potential limitations. This investigation sourced 16 potential reference genes through a comprehensive review of the literature and transcriptome sequencing data analysis. We validated the expression stability of these genes via qRT-PCR across 12 strains, representing the four prevalent serotypes O:3, O:5,27, O:8, and O:9, isolated from diarrheal patient stool samples. This approach aimed to minimize the impact of serotype heterogeneity. After acquiring Cq values, gene stability was evaluated using four established algorithms-ΔCq, geNorm, NormFinder, and BestKeeper-and subsequently synthesized into a consolidated ranking through the Robust Rank Aggregation (RRA) method. Our study suggests that the genes , , , , , and maintain consistent expression across varying culture temperatures, supporting their candidacy as robust housekeeping genes. We advise against the exclusive use of 16S rRNA for this purpose. Should tradition prevail in its utilization, it must be employed with discernment, preferably alongside one or two of the housekeeping genes identified in this study as internal controls.IMPORTANCEIn our study, we focused on identifying stable reference genes for quantitative real-time PCR (qRT-PCR) experiments on cultured at different temperatures (22°C and 37°C). After thoroughly evaluating 16 candidate genes, we identified six genes-, , , , , and -as exhibiting stable expression across these temperature conditions, making them ideal reference genes for studies. This discovery is crucial for ensuring the accuracy and reliability of qRT-PCR data, as the choice of appropriate reference genes is key to normalizing expression data and minimizing experimental variability. Importantly, our research extended beyond bioinformatics analysis by incorporating validation with clinical strains, bridging the gap between theoretical predictions and practical application. This approach not only underscores the robustness and reliability of our findings but also directly addresses the critical need for experimental validation in the field. By providing a set of validated, stably expressed reference genes, our work offers valuable guidance for designing experiments involving , enhancing the reliability of research outcomes, and advancing our understanding of this significant pathogen.