Identification and Validation of Suitable Reference Genes for RT-qPCR Analysis in Mouse Testis Development

Overview

Journal Mol Genet Genomics

Specialty Genetics

Date 2014 Jun 23

PMID 24952483

Citations 27

Authors

Zu-Kang Gong

Shuang-Jie Wang

Yong-Qi Huang

Rui-Qiang Zhao

Qi-Fang Zhu

Wen-Zhen Lin

Affiliations

Soon will be listed here.

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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