Identification and validation of suitable reference genes for RT-qPCR analysis in mouse testis development
Identification and validation of suitable reference genes for RT-qPCR analysis in mouse testis development
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DOI：
10.1007/s00438-014-0877-6
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发表时间：
2014-12-01
影响因子：
3.1
通讯作者：
Lin, Wen-Zhen
中科院分区：
文献类型：
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作者：
Gong, Zu-Kang;Wang, Shuang-Jie;Lin, Wen-Zhen
RT-qPCR是评价基因表达的常用方法;然而，其准确性和可靠性取决于适当参考基因的选择，并且关于可用于不同阶段小鼠睾丸的适当参考基因的可用信息有限。本研究采用RT-qPCR方法，研究了小鼠睾丸中6个不同功能类型的参考基因（Actb、Gapdh、Ppia、Tbp、Rps 29、Hprt 1）在胚胎和生后发育过程中的表达变化。使用五种算法评估推定参考基因的表达稳定性：geNorm、Normandy、Bestkeeper、比较delta C（t）方法和集成工具Referral。结果分析表明，PPIA，Gapdh和Actb被确定为最稳定的基因和几何平均值的PPIA，Gapdh和Actb构成了一个适当的标准化因子的基因表达研究。作为目标测试基因的AT 1的mRNA表达根据哪种参考基因用作内部对照而变化。本研究表明，Ppia，Gapdh和Actb是用于RT-qPCR标准化的6个基因中的合适的参考基因，并在未来的发育中的小鼠睾丸的基因表达的研究中的转录分析提供了重要的信息。
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.