Validation of Suitable Reference Genes for Gene Expression Studies on Yak Testis Development

SIMPLE SUMMARY: Yak (Bos grunniens) provides life materials for herdsmen in high-plateau areas. Improving their low fertility is necessary to meet the demands of the development of the yak industry. The testis is an important organ for male fertility, its development is controlled by a large number of genes. Using real-time reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to explore the quantitative expression of genes can provide insights for illuminating the molecular mechanisms of testis development, but the RT-qPCR data are influenced by the stability of reference genes (RGs). Unfortunately, no available RGs can normalize the gene expression in yak testis development. In this study, the expression stability of 13 candidate genes in yak testis at different developmental stages was evaluated using five different pieces of software. The results showed that the TATA box-binding protein (TBP) and ubiquitously expressed transcript protein (UXT) exhibited high stability across various developmental stages, TBP and hydroxymethylbilane synthase (HMBS) were the most stably expressed genes in immature stages, and mitochondrial ribosomal protein L39 (MRPL39) and TBP exhibited the most stable expression across mature stages. This study provided suitable RGs for gene expression studies in yak testis development. ABSTRACT: Testis has an important function in male reproduction. Its development is regulated by a large number of genes. The real-time reserve transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a useful tool to evaluate the gene expression levels. However, unsuitable reference genes (RGs) can cause the misinterpretation of gene expression levels. Unfortunately, the ideal RGs for yak testis development are yet to be studied. In this study, 13 commonly used RGs were selected to identify the most stable RGs in yak testis at four different developmental stages, including two immature stages (6 months and 18 months) and two mature stages (30 months and 6 years). This study used GeNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder programs to evaluate the stability of 13 candidate genes. The results of RefFinder showed that the stabilities of TATA box-binding protein (TBP) and ubiquitously expressed transcript protein (UXT) were ranked the top two across all developmental stages. TBP and hydroxymethylbilane synthase (HMBS) were stably expressed in immature stages, while mitochondrial ribosomal protein L39 (MRPL39) and TBP had higher stability than other candidate genes in mature stages. This study provided valuable information for gene expression studies to assist further investigation on the molecular mechanisms in underlying yak testis development.