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Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms

Real-time quantitative PCR (RT-qPCR) is a widely applied technique for relative quantification of gene expression. In this context, the selection of a suitable reference gene (RG) is an essential step for obtaining reliable and biologically relevant RT-qPCR results. The present study aimed to determ...

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Autores principales: Ma, Jianfeng, Chen, Jingyun, Gan, Mailin, Chen, Lei, Zhao, Ye, Niu, Lili, Zhu, Yan, Zhang, Shunhua, Li, Xuewei, Guo, Zongyi, Wang, Jinyong, Zhu, Li, Shen, Linyuan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9583855/
https://www.ncbi.nlm.nih.gov/pubmed/36275473
http://dx.doi.org/10.7717/peerj.14221
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author Ma, Jianfeng
Chen, Jingyun
Gan, Mailin
Chen, Lei
Zhao, Ye
Niu, Lili
Zhu, Yan
Zhang, Shunhua
Li, Xuewei
Guo, Zongyi
Wang, Jinyong
Zhu, Li
Shen, Linyuan
author_facet Ma, Jianfeng
Chen, Jingyun
Gan, Mailin
Chen, Lei
Zhao, Ye
Niu, Lili
Zhu, Yan
Zhang, Shunhua
Li, Xuewei
Guo, Zongyi
Wang, Jinyong
Zhu, Li
Shen, Linyuan
author_sort Ma, Jianfeng
collection PubMed
description Real-time quantitative PCR (RT-qPCR) is a widely applied technique for relative quantification of gene expression. In this context, the selection of a suitable reference gene (RG) is an essential step for obtaining reliable and biologically relevant RT-qPCR results. The present study aimed to determine the expression stability of commonly used RGs in mouse skeletal muscle tissue. The expression pattern of eight RGs (ACTB, GAPDH, HPRT, YWHAZ, B2M, PPIA, TUBA and 18S) were evaluated by RT-qPCR in different sample groups classified based on genetic background, muscle tissue type, and growth stage, as well as in a C2C12 myoblast cell line model. Five computational programs were included in the study (comparative ΔCq value, NormFinder, BestKeeper, geNorm, RefFinder) to evaluate the expression stability of RGs. Furthermore, the normalization effects of RGs in soleus (SOL) and gastrocnemius (GAS) muscle tissue were evaluated. Collectively, ACTB, HPRT and YWHAZ were shown to be the most stable RGs, while GADPH and 18S were the least stable. Therefore, the combined use of ACTB, HPRT and YWHAZ is recommended for the normalization of gene expression results in experiments with murine skeletal muscle. The results discussed herein provide a foundation for gene expression analysis by RT-qPCR in mammalian skeletal muscle.
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spelling pubmed-95838552022-10-21 Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms Ma, Jianfeng Chen, Jingyun Gan, Mailin Chen, Lei Zhao, Ye Niu, Lili Zhu, Yan Zhang, Shunhua Li, Xuewei Guo, Zongyi Wang, Jinyong Zhu, Li Shen, Linyuan PeerJ Biochemistry Real-time quantitative PCR (RT-qPCR) is a widely applied technique for relative quantification of gene expression. In this context, the selection of a suitable reference gene (RG) is an essential step for obtaining reliable and biologically relevant RT-qPCR results. The present study aimed to determine the expression stability of commonly used RGs in mouse skeletal muscle tissue. The expression pattern of eight RGs (ACTB, GAPDH, HPRT, YWHAZ, B2M, PPIA, TUBA and 18S) were evaluated by RT-qPCR in different sample groups classified based on genetic background, muscle tissue type, and growth stage, as well as in a C2C12 myoblast cell line model. Five computational programs were included in the study (comparative ΔCq value, NormFinder, BestKeeper, geNorm, RefFinder) to evaluate the expression stability of RGs. Furthermore, the normalization effects of RGs in soleus (SOL) and gastrocnemius (GAS) muscle tissue were evaluated. Collectively, ACTB, HPRT and YWHAZ were shown to be the most stable RGs, while GADPH and 18S were the least stable. Therefore, the combined use of ACTB, HPRT and YWHAZ is recommended for the normalization of gene expression results in experiments with murine skeletal muscle. The results discussed herein provide a foundation for gene expression analysis by RT-qPCR in mammalian skeletal muscle. PeerJ Inc. 2022-10-17 /pmc/articles/PMC9583855/ /pubmed/36275473 http://dx.doi.org/10.7717/peerj.14221 Text en ©2022 Ma et al. https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Biochemistry
Ma, Jianfeng
Chen, Jingyun
Gan, Mailin
Chen, Lei
Zhao, Ye
Niu, Lili
Zhu, Yan
Zhang, Shunhua
Li, Xuewei
Guo, Zongyi
Wang, Jinyong
Zhu, Li
Shen, Linyuan
Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms
title Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms
title_full Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms
title_fullStr Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms
title_full_unstemmed Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms
title_short Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms
title_sort comparison of reference gene expression stability in mouse skeletal muscle via five algorithms
topic Biochemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9583855/
https://www.ncbi.nlm.nih.gov/pubmed/36275473
http://dx.doi.org/10.7717/peerj.14221
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