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Identification and validation of suitable reference genes for RT-qPCR analysis in mouse testis development

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Abstract

RT-qPCR is a commonly used method for evaluating gene expression; however, its accuracy and reliability are dependent upon the choice of appropriate reference gene(s), and there is limited information available on suitable reference gene(s) that can be used in mouse testis at different stages. In this study, using the RT-qPCR method, we investigated the expression variations of six reference genes representing different functional classes (Actb, Gapdh, Ppia, Tbp, Rps29, Hprt1) in mice testis during embryonic and postnatal development. The expression stabilities of putative reference genes were evaluated using five algorithms: geNorm, NormFinder, Bestkeeper, the comparative delta C t method and integrated tool RefFinder. Analysis of the results showed that Ppia, Gapdh and Actb were identified as the most stable genes and the geometric mean of Ppia, Gapdh and Actb constitutes an appropriate normalization factor for gene expression studies. The mRNA expression of AT1 as a test gene of interest varied depending upon which of the reference gene(s) was used as an internal control(s). This study suggested that Ppia, Gapdh and Actb are suitable reference genes among the six genes used for RT-qPCR normalization and provide crucial information for transcriptional analyses in future studies of gene expression in the developing mouse testis.

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Acknowledgments

This research project was funded by the National Natural Science Foundation of China (Grant No. 81160263), the Scientific Research Fund of Guangxi Education Department (Grant No. 200103YB025) and the Guangxi Natural Science Foundation Program (Grant No. 2010GXNSFA013162). We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

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Authors and Affiliations

  1. Guangxi Nanning Blood Center, Nanning, 530007, Guangxi, China

    Zu-Kang Gong

  2. Department of Biochemistry and Molecular Biology, School of Preclinical Sciences, Guangxi Medical University, Nanning, 530021, Guangxi, China

    Shuang-Jie Wang, Yong-Qi Huang, Rui-Qiang Zhao, Qi-Fang Zhu & Wen-Zhen Lin

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  1. Zu-Kang Gong
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  2. Shuang-Jie Wang
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  3. Yong-Qi Huang
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Correspondence to Wen-Zhen Lin.

Additional information

Communicated by S. Hohmann.

Z.-K. Gong and S.-J. Wang contributed equally to this work and should be considered as co-first authors.

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Gong, ZK., Wang, SJ., Huang, YQ. et al. Identification and validation of suitable reference genes for RT-qPCR analysis in mouse testis development. Mol Genet Genomics 289, 1157–1169 (2014). https://doi.org/10.1007/s00438-014-0877-6

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  • DOI: https://doi.org/10.1007/s00438-014-0877-6

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