Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in .

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 .