The Selection of Reference Genes for Quantitative Real-Time PCR in the Ashidan Yak Mammary Gland During Lactation and Dry Period

Abstract

Investigating the critical genes related to milk synthesis is essential for the improvement of the milk yield of the yak. Real-time quantitative polymerase chain reaction (RT-qPCR) is a reliable and widely used method to measure and evaluate gene expression levels. Selection of suitable reference genes is mandatory to acquire accurate normalization of gene expression results from RT-qPCR. To select the most stable reference genes for reliable normalization of mRNA expression by RT-qPCR in the mammary gland of the Ashidan yak, we selected 16 candidate reference genes and analyzed their expression stability at different physiological stages (lactation and dry period). The expression stability of the candidate reference genes was assessed using geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder methods. The results showed that the hydroxymethylbilane synthase gene (HMBS) and the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) were the most stable genes across all treatment samples. The reliability of selected reference genes was validated by normalizing relative expression of the lactation-related 60S ribosomal protein L35 gene (RPL35). The relative expression of RPL35 varied considerably according to the different reference genes. This work provides valuable information to further promote research in the molecular mechanisms involved in lactation and mammary gland development and provides a foundation for the improvement of the milk yield and quality of the Ashidan yak.