Selection and validation of reference genes for RT-qPCR in adipose and longissimus dorsi muscle tissues of buffalo
Selection and validation of reference genes for RT-qPCR in adipose and longissimus dorsi muscle tissues of buffalo
复制标题
水牛脂肪和背最长肌组织中 RT-qPCR 内参基因的选择和验证
DOI：
10.1080/10495398.2020.1811715
复制
发表时间：
2020-08-28
影响因子：
3.7
通讯作者：
Huang, Jieping
中科院分区：
文献类型：
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作者：
Feng, Xue;Cao, Xiaodan;Huang, Jieping
摘要实时定量PCR（RT-qPCR）是一种广泛应用于基因表达检测和评价的方法。RT-qPCR的精确性和可靠性关键取决于合适的参考基因（RGs）的选择。在这项研究中，作出了努力，以确定最佳的RGs的RT-qPCR分析的脂肪和背最长肌（LM）在水牛。首先对RNA测序数据进行分析，获得10个在脂肪组织中高表达且稳定的候选基因（FKBP 1A、C25 H16 orf 72、PNRC 2、IQGAP 1、ATP 5 PD、RPL 6、NDUFB 4、TRA 2A、CAPRIN 1和METAP 2）。还选择了四个其他鉴定的RG（GAPDH、ACTB、TOP 2B和UXT）。通过三种算法（geNorm、Normalization和BestKeeper）评估候选RG的表达稳定性，然后通过脂肪细胞和肌细胞标志物进一步验证。我们的结果表明，UXT和TOP 2B是水牛脂肪组织RT-qPCR分析的最佳RG; RPL 6、UXT和TOP 2B三个RG是水牛脂肪和LM组织RT-qPCR分析的最佳RG。该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。
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.