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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.

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