Abstract
Background:
Yersinia enterocolitica, a species within the genus Yersinia, thrives optimally at 22–25°C but can also grow at mammalian core body temperature of 37°C. This dual temperature adaptability necessitates the establishment of both temperature conditions in research to examine the effects on various biological inquiries. In quantitative real-time PCR (qRT-PCR) assays, the selection of appropriate housekeeping genes is vital for data accuracy. Nevertheless, the paucity of alternatives and information frequently leads to the default use of the 16s rRNA gene, despite potential limitations.
Methodology:
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 Y. enterocolitica 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. Post-acquisition of Cq values, gene stability was evaluated using four established algorithms—∆Cq, geNorm, NormFinder, and BestKeeper—and subsequently synthesized into a consolidated ranking through the Robust Rank Aggregation (RRA) method.
Conclusion:
Our study suggests that the genes glnS, nuoB, glmS, gyrB, dnaK, and thrS maintain consistent expression across varying culture temperatures, endorsing them as robust housekeeping gene candidates. 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.