College of Life Sciences, Xinyang Normal University, Xinyang, China.
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.
Anim Biotechnol. 2022 Jun;33(3):526-535. doi: 10.1080/10495398.2020.1811715. Epub 2020 Aug 31.
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 (, , , , , , , , , and ) that with high and stable expression across adipose tissues. Four other identified RGs (, , , and ) 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 and were the optimal RGs for RT-qPCR analysis across adipose tissues in buffaloes; three RGs, , , and , 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.
实时荧光定量 PCR（RT-qPCR）被广泛用于测量和评估基因表达。RT-qPCR 的精度和可靠性严重依赖于合适的参考基因（RGs）的选择。在这项研究中，我们努力确定水牛脂肪和背最长肌（LM）中 RT-qPCR 分析的最佳 RG。首先对 RNA 测序数据进行分析，以获得 10 个候选基因（、、、、、、、、和），这些基因在脂肪组织中具有高且稳定的表达。还选择了另外 4 个鉴定的 RG（、、、和）。候选 RG 的表达稳定性通过三种算法（geNorm、NormFinder 和 BestKeeper）进行评估，然后用脂肪细胞和肌细胞标记物进一步验证。我们的结果表明，和是水牛脂肪组织中 RT-qPCR 分析的最佳 RG；三个 RG、、和，是水牛脂肪和 LM 组织中 RT-qPCR 分析的最佳 RG。这项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。
