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

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 th...

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Bibliographic Details
Published in:Molecular genetics and genomics : MGG 2014-12, Vol.289 (6), p.1157-1169
Main Authors: Gong, Zu-Kang, Wang, Shuang-Jie, Huang, Yong-Qi, Zhao, Rui-Qiang, Zhu, Qi-Fang, Lin, Wen-Zhen
Format: Article
Language:English
Subjects:
Algorithms
Animal Genetics and Genomics
Animals
Biochemistry
Biomedical and Life Sciences
Cell division
DNA Primers
Gene Expression
genes
Genomics
Human Genetics
Infertility
Laboratory animals
Life Sciences
Male
Medical research
messenger RNA
Mice
Microbial Genetics and Genomics
Original Paper
Plant Genetics and Genomics
postnatal development
Real-Time Polymerase Chain Reaction - standards
Receptor, Angiotensin, Type 1 - genetics
Reference Standards
Spermatogenesis
testes
Testis - embryology
Testis - growth & development
Testis - metabolism
transcription (genetics)
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Summary: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 Cₜmethod 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.
ISSN:1617-4615
1617-4623
DOI:10.1007/s00438-014-0877-6