Selection of reference genes for gene expression studies related to intramuscular fat deposition in Capra hircus skeletal muscle

PLoS One. 2015 Mar 20;10(3):e0121280. doi: 10.1371/journal.pone.0121280. eCollection 2015.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adiposity / genetics*
  • Animals
  • DNA Primers / metabolism
  • Gene Expression Profiling
  • Gene Expression Regulation, Developmental*
  • Genetic Association Studies
  • Goats / genetics*
  • Male
  • Muscle, Skeletal / growth & development
  • Muscle, Skeletal / metabolism*
  • PPAR gamma / genetics
  • PPAR gamma / metabolism
  • Reference Standards
  • Reproducibility of Results
  • Software


  • DNA Primers
  • PPAR gamma

Grant support

The work was supported by the "twelfth Five-Year" Key Projects of Animal and Poultry Breeding Research of Sichuan Province (No. 2011NZ0099-36), National High Technology Research and Development Program of China (No. 2012BAD13B06), the Key Demonstration Project of Science and Technology Innovation Industrial Chain in Sichuan province (No. 2011NZ0003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.