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Thirteen reference genes were investigated to determine their stability to be used as a housekeeping in gene expression studies in skeletal muscle of chickens. Five different algorithms were used for ranking of reference genes and results suggested that individual rankings of the genes differed among them. The stability of the expression of reference genes were validated using samples obtained from the  Pectoralis major  muscle in chicken. Samples were obtained from chickens in different development periods post hatch and under different nutritional diets. For gene expression calculation the ΔΔCt approach was applied to compare relative expression of pairs of genes within each of 52 samples when normalized to  mitochondrially encoded cytochrome c oxidase II  (MT-CO2) target gene. Our findings showed that  hydroxymethylbilane synthase  (HMBS) and  hypoxanthine phosphoribosyl transferase 1  (HPRT1) are the most stable reference genes while  transferrin receptor  (TFRC) and  beta-2-microglobulin  (B2M) ranked as the least stable genes in the  Pectoralis major  muscle of chickens. Moreover, our results revealed that HMBS and HPRT1 gene expression did not change due to dietary variations and thus it is recommended for accurate normalization of RT-qPCR data in chicken  Pectoralis major  muscle.

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Identification of Suitable Reference Genes for Real Time Quantitative Polymerase Chain Reaction Assays on Pectoralis major Muscle in Chicken (Gallus gallus )