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Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae
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 refere...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9355380/ https://www.ncbi.nlm.nih.gov/pubmed/35937288 http://dx.doi.org/10.3389/fvets.2022.934907 |
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author | Li, Shiyang Zhou, Yanqing Yuan, Ting Feng, Zhixin Zhang, Zhenzhen Wu, Yuzi Xie, Qingyun Wang, Jia Li, Quan Deng, Zhibang Yu, Yanfei Yuan, Xiaomin |
author_facet | Li, Shiyang Zhou, Yanqing Yuan, Ting Feng, Zhixin Zhang, Zhenzhen Wu, Yuzi Xie, Qingyun Wang, Jia Li, Quan Deng, Zhibang Yu, Yanfei Yuan, Xiaomin |
author_sort | Li, Shiyang |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9355380 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-93553802022-08-06 Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae Li, Shiyang Zhou, Yanqing Yuan, Ting Feng, Zhixin Zhang, Zhenzhen Wu, Yuzi Xie, Qingyun Wang, Jia Li, Quan Deng, Zhibang Yu, Yanfei Yuan, Xiaomin Front Vet Sci Veterinary Science 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. Frontiers Media S.A. 2022-07-22 /pmc/articles/PMC9355380/ /pubmed/35937288 http://dx.doi.org/10.3389/fvets.2022.934907 Text en Copyright © 2022 Li, Zhou, Yuan, Feng, Zhang, Wu, Xie, Wang, Li, Deng, Yu and Yuan. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Veterinary Science Li, Shiyang Zhou, Yanqing Yuan, Ting Feng, Zhixin Zhang, Zhenzhen Wu, Yuzi Xie, Qingyun Wang, Jia Li, Quan Deng, Zhibang Yu, Yanfei Yuan, Xiaomin Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae |
title | Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae |
title_full | Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae |
title_fullStr | Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae |
title_full_unstemmed | Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae |
title_short | Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae |
title_sort | selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in mycoplasma hyopneumoniae |
topic | Veterinary Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9355380/ https://www.ncbi.nlm.nih.gov/pubmed/35937288 http://dx.doi.org/10.3389/fvets.2022.934907 |
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