Transcriptome-Based Evaluation of Optimal Reference Genes for Quantitative Real-Time PCR in Yak Stomach throughout the Growth Cycle

SIMPLE SUMMARY: The stomach is one of the primary sites for the digestion and absorption of nutrients. Quantifying related gene expression patterns using quantitative real-time PCR (RT-qPCR) is conducive to further understanding the molecular mechanisms underlying nutrition metabolism in the yak stomach. The authenticity of RT-qPCR data is affected by the selection of reference genes. Unfortunately, no studies have demonstrated suitable reference genes for the normalization of RT-qPCR data in the yak stomach. In this study, 15 candidate reference genes (CRGs) were identified according to transcriptome sequencing (RNA-seq) results and the previous literature. Five algorithms were used to evaluate the stability of the CRGs across the entire developmental stage in the yak stomach. RPS15, MRPL39, and RPS23 were found to be the most stable genes in the yak stomach from birth to adulthood. This study indicates the appropriate reference genes for gene expression analysis via RT-qPCR in the yak stomach. ABSTRACT: Efficient nutritional assimilation and energy metabolism in the stomachs of yaks contribute to their adaption to harsh environments. Accurate gene expression profile analysis will help further reveal the molecular mechanism of nutrient and energy metabolism in the yak stomach. RT-qPCR is regarded as an accurate and dependable method for analyzing gene expression. The selection of reference genes is essential to obtain meaningful RT-qPCR results, especially in longitudinal gene expression studies of tissues and organs. Our objective was to select and validate optimal reference genes from across the transcriptome as internal controls for longitudinal gene expression studies in the yak stomach. In this study, 15 candidate reference genes (CRGs) were determined according to transcriptome sequencing (RNA-seq) results and the previous literature. The expression levels of these 15 CRGs were quantified using RT-qPCR in the yak stomach, including the rumen, reticulum, omasum and abomasum at five stages: 0 days, 20 days, 60 days, 15 months and three years old (adult). Subsequently, the expression stabilities of these 15 CRGs were evaluated via four algorithms: geNorm, NormFinder, BestKeeper and the comparative C(T) method. Furthermore, RefFinder was employed to obtain a comprehensive ranking of the stability of CRGs. The analysis results indicate that RPS15, MRPL39 and RPS23 are the most stable genes in the yak stomach throughout the growth cycle. In addition, to verify the reliability of the selected CRGs, the relative expression levels of HMGCS2 were quantified via RT-qPCR using the three most stable or the three least stable CRGs. Overall, we recommend combining RPS15, MRPL39 and RPS23 as reference genes for the normalization of RT-qPCR data in the yak stomach throughout the growth cycle.