Evaluation of Reference Gene Stability in Goat Skeletal Muscle Satellite Cells during Proliferation and Differentiation Phases

TL;DR

This study identifies the best reference genes for accurate gene expression analysis in goat muscle cells during growth and development.

Contribution

The study provides a validated set of optimal reference genes for RT-qPCR in goat skeletal muscle satellite cells.

Findings

RPL14 and RPS15A were the most stable reference genes during proliferation and differentiation.

Traditional reference genes like ACTB and GAPDH showed high variability and were unsuitable.

The validated reference genes can improve accuracy in gene expression studies of goat muscle cells.

Abstract

Investigating the proliferation and differentiation of skeletal muscle satellite cells (MuSCs) in vitro is crucial for elucidating the underlying mechanisms of skeletal muscle development in goats. Real-time quantitative PCR (RT-qPCR) is a widely utilized technique for quantifying the expression levels of target genes. A significant challenge associated with this method is the identification of optimal reference genes for accurate normalization. In this study, we evaluated ten candidate reference genes for the standardization of gene expression. Our results indicated that RPL14 and RPS15A constituted the most stable reference gene combination during the proliferation and differentiation of goat MuSCs.

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.

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