2. Academic Validation
  3. Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae

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

  • Front Vet Sci. 2022 Jul 22:9:934907. doi: 10.3389/fvets.2022.934907.
Shiyang Li 1 2,  Yanqing Zhou 2 3 4,  Ting Yuan 2,  Zhixin Feng 2 3,  Zhenzhen Zhang 2,  Yuzi Wu 2,  Qingyun Xie 2,  Jia Wang 2,  Quan Li 5,  Zhibang Deng 1,  Yanfei Yu 1 2 3 6,  Xiaomin Yuan 1
Affiliations

Affiliations

  • 1 College of Veterinary Medicine, Hunan Agricultural University, Changsha, China.
  • 2 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.
  • 3 College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
  • 4 Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, China.
  • 5 College of Veterinary Medicine, Yangzhou University, Yangzhou, China.
  • 6 School of Food and Biological Engineering, Jiangsu University, Zhenjiang, China.
PMID: 35937288 DOI: 10.3389/fvets.2022.934907
Abstract

Mycoplasma hyopneumoniae 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 M. hyopneumoniae have been reported. The aim of this study was to screen the most stable genes for RT-qPCR analysis in M. hyopneumoniae under different conditions. Therefore, a total of 13 candidate internal reference genes (rpoC, Lipo, sgaB, oppB, hypo621, oppF, gyrB, uvrA, P146, prfA, proS, gatB, and hypo499) of M. hyopneumoniae filtered according to the reported quantitative proteomic analysis and the 16S rRNA 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 gatB gene was the most suitable internal reference gene for samples of the different virulence strains in different growth phases for M. hyopneumoniae, followed by prfA, hypo499, and gyrB.

Keywords

Mycoplasma hyopneumoniae; RT-qPCR – real-time quantitative polymerase chain reaction; gatB; internal reference genes; virulence.

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