Abstract

Real-time quantitative PCR (RT-qPCR) is widely used to measure and evaluate gene expression. The precision and reliability of RT-qPCR are critically dependent on the selection of suitable reference genes (RGs). In this study, an effort was made to identify the optimal RGs for RT-qPCR analysis of adipose and the longissimus dorsi muscle (LM) in buffaloes. RNA sequencing data were firstly analyzed to obtain 10 candidate genes (FKBP1A, C25H16orf72, PNRC2, IQGAP1, ATP5PD, RPL6, NDUFB4, TRA2A, CAPRIN1, and METAP2) that with high and stable expression across adipose tissues. Four other identified RGs (GAPDH, ACTB, TOP2B, and UXT) were selected as well. The expression stability of the candidate RGs was evaluated by three algorithms (geNorm, NormFinder, and BestKeeper) and then further validated by adipocyte and myocyte markers. Our results showed that UXT and TOP2B were the optimal RGs for RT-qPCR analysis across adipose tissues in buffaloes; three RGs, RPL6, UXT, and TOP2B, were the optimal RGs for RT-qPCR analysis across adipose and the LM tissues in buffaloes. This study provides significant information for improving the accuracy of gene expression in research on intramuscular fat deposition in buffaloes.

摘要

实时定量 PCR (RT-qPCR) 广泛用于测量和评估基因表达。RT-qPCR 的精确度和可靠性严重依赖于选择合适的参考基因 (RGs)。在这项研究中，努力确定用于 RT-qPCR 分析水牛脂肪和背最长肌 (LM) 的最佳 RG。首先对RNA测序数据进行分析，得到10个候选基因（FKBP1A、C25H16orf72、PNRC2、IQGAP1、ATP5PD、RPL6、NDUFB4、TRA2A、CAPRIN1和METAP2) 在脂肪组织中具有高而稳定的表达。还选择了其他四个已识别的 RG（GAPDH、ACTB、TOP2B和UXT）。通过三种算法（geNorm、NormFinder 和 BestKeeper）评估候选 RGs 的表达稳定性，然后通过脂肪细胞和肌细胞标记物进一步验证。我们的结果表明，UXT和TOP2B是水牛脂肪组织 RT-qPCR 分析的最佳 RG；三个 RG，RPL6、UXT和TOP2B，是在水牛的脂肪和 LM 组织中进行 RT-qPCR 分析的最佳 RG。本研究为提高水牛肌内脂肪沉积研究中基因表达的准确性提供了重要信息。