Abstract
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.
Highlights
Meat from goats is becoming more widely accepted around the world due to the increasing demand for sustainable foods and the low cholesterol content and high nutritive value of goat meat [1]
There is an increasing number of reports demonstrating that the expression of most reference genes–including some commonly used reference genes such as GAPDH, ACTB and 18S–is context dependent [5,11], as their expression may vary significantly depending on the experimental conditions being investigated [12]
Given the underlying issues associated with the use of non-validated reference genes for data normalization, in this study, we aimed to identify a set of reference genes that could serve as proper reference genes in Capra hircus skeletal muscle
Summary
Meat from goats is becoming more widely accepted around the world due to the increasing demand for sustainable foods and the low cholesterol content and high nutritive value of goat meat [1]. QPCR requires data normalization to correct for variability in the amount and quality of starting material, RNA stability, content, enzymatic efficiencies, and technique in the qPCR experimental process [5]. To address these problems, several strategies have been implemented for data normalization including quantifying RNA input or number of cells used. The amount of RNA maybe insufficient at times and accurate computation of cell number is often infeasible Another proposed strategy is to use reference genes in data normalization; this strategy is currently considered a robust approach in most cases [8]. The validity of candidate reference genes has to be well established to circumvent problems in data normalization
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