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

作者：Chuchu Li, Lü Zhou, Xiaoxuan Ma, Liguo Zhu, Jia Li, Lingning Meng, Mei Han, Danwei Wang, Han Shen, Chang Liu · 发表于：Microbiology Spectrum · 年份：2024 · DOI：10.1128/spectrum.01146-24 · 被引用次数：3 · 研究领域：Molecular Biology Techniques and Applications、Yersinia bacterium, plague, ectoparasites research、Bacterial Identification and Susceptibility Testing

ABSTRACT Yersinia enterocolitica , a species within the genus Yersinia , 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 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. 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 glnS , nuoB , glmS , gyrB , dnaK , and thrS maintain consistent expression across varying culture temperatures, supporting their candidacy as robust housekeeping genes. We advise against the exclusive use ...