Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen

SIMPLE SUMMARY: The rumen plays an essential role as a digestive organ and serves as the primary site of energy substrate absorption for the productive ruminants. Understanding gene expression profiles is necessary to explore the intrinsic regulatory mechanisms of rumen development in goats. The selection of suitable reference genes (RGs) was the primary assay before the real-time quantitative PCR (RT-qPCR). We identified sixteen genome-wide candidate RGs for normalization of gene expression assessments in goat rumen tissues. We demonstrate that the RGs selected (RPS4X and RPS6) were more stably expressed than the commonly used HKGs (ACTB and GAPDH) in goat rumen tissues, suggesting that the ribosomal protein gene family may be another source for the RG pool. ABSTRACT: As the largest chamber of the ruminant stomach, the rumen not only serves as the principal absorptive surface and nutrient transport pathway from the lumen into the animal, but also plays an important short-chain fatty acid (SCFA) metabolic role in addition to protective functions. Accurate characterization of the gene expression profiles of genes of interest is essential to the exploration of the intrinsic regulatory mechanisms of rumen development in goats. Thus, the selection of suitable reference genes (RGs) is an important prerequisite for real-time quantitative PCR (RT-qPCR). In the present study, 16 candidate RGs were identified from our previous transcriptome sequencing of caprine rumen tissues. The quantitative expressions of the candidate RGs were measured using the RT-qPCR method, and the expression stability of the RGs was assessed using the geNorm, NormFinder, and BestKeeper programs. GeNorm analysis showed that the M values were less than 0.5 for all the RGs except GAPT4, indicating that they were stably expressed in the rumen tissues throughout development. RPS4X and RPS6 were the two most stable RGs. Furthermore, the expressions of two randomly selected target genes (IGF1 and TOP2A), normalized by the selected most stable RGs (RPS4X and RPS6), were consistent with the results of RNA sequencing, while the use of GAPDH and ACTB as RGs resulted in altered profiles. Overall, RPS4X and RPS6 showed the highest expression stability and the lowest coefficients of variation, and could be used as the optimal reference combination for quantifying gene expression in rumen tissues via RT-qPCR analysis.