Li Shiyang, Zhou Yanqing, Yuan Ting, Feng Zhixin, Zhang Zhenzhen, Wu Yuzi, Xie Qingyun, Wang Jia, Li Quan, Deng Zhibang, Yu Yanfei, Yuan Xiaomin
College of Veterinary Medicine, Hunan Agricultural University, Changsha, China.
Key Laboratory of Veterinary Biological Engineering and Technology, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Ministry of Agriculture and Rural Affairs, Nanjing, China.
Front Vet Sci. 2022 Jul 22;9:934907. doi: 10.3389/fvets.2022.934907. eCollection 2022.
is the etiological agent of swine enzootic pneumonia (EP), which resulting in considerable economic losses in pig farming globally. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a major tool for gene expression studies. However, no internal reference genes for normalization of RT-qPCR data of have been reported. The aim of this study was to screen the most stable genes for RT-qPCR analysis in under different conditions. Therefore, a total of 13 candidate internal reference genes (, and ) of filtered according to the reported quantitative proteomic analysis and the internal reference gene frequently used in other bacteria were selected for RT-qPCR analysis. The mRNAs from different virulence strains (168, 168 L, J, NJ, and LH) at five different growth phases were extracted. The corresponding cycle threshold (Ct) values of the 25 reverse transcribed cDNAs using the 14 candidate genes were determined. Different internal reference genes or combinations were then screened for expression stability analysis using various statistical tools and algorithms, including geNorm, BestKeeper, and NormFinder software, to ensure the reliability of the analysis. Through further comprehensive evaluation of the RefFinder software, it is concluded that the gene was the most suitable internal reference gene for samples of the different virulence strains in different growth phases for , followed by , and .
是猪地方流行性肺炎（EP）的病原体，在全球养猪业中造成了相当大的经济损失。逆转录定量聚合酶链反应（RT-qPCR）是基因表达研究的主要工具。然而，尚未有关于用于标准化其RT-qPCR数据的内参基因的报道。本研究的目的是筛选在不同条件下用于其RT-qPCR分析的最稳定基因。因此，根据已报道的定量蛋白质组学分析筛选出其13个候选内参基因（、和），并选择其他细菌中常用的内参基因进行RT-qPCR分析。提取了来自不同毒力菌株（168、168L、J、NJ和LH）在五个不同生长阶段的mRNA。使用这14个候选基因测定了25个逆转录cDNA的相应循环阈值（Ct）值。然后使用包括geNorm、BestKeeper和NormFinder软件在内的各种统计工具和算法筛选不同的内参基因或组合进行表达稳定性分析，以确保分析的可靠性。通过RefFinder软件的进一步综合评估，得出基因是不同毒力菌株在不同生长阶段样本中最适合的内参基因，其次是和。
