Zhan Siyuan, Zhang Lufei, Zhong Tao, Wang Linjie, Guo Jiazhong, Cao Jiaxue, Li Li, Zhang Hongping
Key Laboratory of Livestock and Poultry Multi-Omics, Ministry of Agriculture and Rural Affairs, College of Animal and Technology, Sichuan Agricultural University, Chengdu 611130, China.
Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China.
Animals (Basel). 2024 Aug 26;14(17):2479. doi: 10.3390/ani14172479.
The process of skeletal muscle development is intricate and involves the regulation of a diverse array of genes. Accurate gene expression profiles are crucial for studying muscle development, making it essential to choose the right reference genes for real-time quantitative PCR (RT-qPCR). In the present study, eight candidate reference genes were identified from our previous transcriptome sequencing analysis of caprine skeletal muscle satellite cells (MuSCs), and two traditional reference genes (ACTB and GAPDH) were assessed. The quantitative levels of the candidate reference genes were determined through the RT-qPCR technique, while the stability of their expression was evaluated utilizing the GeNorm, NormFinder, BestKeeper, and RefFinder programs. Furthermore, the chosen reference genes were utilized for the normalization of the gene expression levels of PCNA and Myf5. It was determined that conventional reference genes, including ACTB and GAPDH, were not appropriate for normalizing target gene expression. Conversely, RPL14 and RPS15A, identified through RNA sequencing analysis, exhibited minimal variability and were identified as the optimal reference genes for normalizing gene expression during the proliferation and differentiation of goat MuSCs. Our research offers a validated panel of optimal reference genes for the detection of differentially expressed genes in goat muscle satellite cells using RT-qPCR.
骨骼肌发育过程错综复杂,涉及多种基因的调控。准确的基因表达谱对于研究肌肉发育至关重要,这使得为实时定量PCR(RT-qPCR)选择合适的内参基因必不可少。在本研究中,从我们之前对山羊骨骼肌卫星细胞(MuSCs)的转录组测序分析中鉴定出八个候选内参基因,并评估了两个传统内参基因(ACTB和GAPDH)。通过RT-qPCR技术测定候选内参基因的定量水平,同时利用GeNorm、NormFinder、BestKeeper和RefFinder程序评估其表达稳定性。此外,所选内参基因用于标准化PCNA和Myf5的基因表达水平。结果表明,包括ACTB和GAPDH在内的传统内参基因不适用于标准化靶基因表达。相反,通过RNA测序分析鉴定出的RPL14和RPS15A表现出最小的变异性,被确定为在山羊MuSCs增殖和分化过程中标准化基因表达的最佳内参基因。我们的研究为使用RT-qPCR检测山羊肌肉卫星细胞中差异表达基因提供了一组经过验证的最佳内参基因。