Selection of Reference Genes for Gene Expression Studies Related to Intramuscular Fat Deposition in Capra hircus Skeletal Muscle

The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.

筛选合适的内参基因对于通过实时定量聚合酶链反应（quantitative real-time PCR，qPCR）开展基因表达研究并获得可靠结果至关重要，因为内参基因的表达在不同实验条件下可能存在显著差异。然而在多数情况下，研究人员在进行数据标准化时往往直接使用常用内参基因，却未开展恰当验证，这可能导致数据解读出现偏差。本研究旨在筛选一套最优内参基因，用于精准标准化发育过程中与肌内脂肪（intramuscular fat，IMF）沉积相关的基因表达分析。本研究针对两种肌肉组织（背最长肌与股二头肌）在不同发育阶段的样本，采用geNorm、NormFinder和BestKeeper三种不同算法，对8种内参基因（PPIB、HMBS、RPLP0、B2M、YWHAZ、18S、GAPDH及ACTB）的表达稳定性进行了评估。三种算法得出的结果基本一致。研究发现PPIB与HMBS是稳定性最优的内参基因，而常用的18S和GAPDH则是表达稳定性最差的内参基因，其表达水平在不同发育阶段存在显著波动。此外，为阐明合适内参基因对获得可靠研究结果的关键作用，本研究分别以稳定性最优与最差的内参基因作为标准化参照，对PPARG的表达水平进行了分析。以稳定性最优内参基因标准化得到的PPARG相对表达水平，与以稳定性最差内参基因标准化得到的结果存在显著差异。因此，在使用内参基因前，必须针对特定实验条件对其稳定性进行评估验证。综上，PPIB与HMBS是开展发育过程中骨骼肌肌内脂肪沉积相关基因表达分析的最优内参基因。