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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 )

Author

Listed:
  • Carlos S Nascimento
  • Leandro T Barbosa
  • Claudson Brito
  • Roberta P M Fernandes
  • Renata S Mann
  • Ana Paula G Pinto
  • Haniel C Oliveira
  • Mike V Dodson
  • Simone E F Guimarães
  • Marcio S Duarte

Abstract

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.

Suggested Citation

  • Carlos S Nascimento & Leandro T Barbosa & Claudson Brito & Roberta P M Fernandes & Renata S Mann & Ana Paula G Pinto & Haniel C Oliveira & Mike V Dodson & Simone E F Guimarães & Marcio S Duarte, 2015. "Identification of Suitable Reference Genes for Real Time Quantitative Polymerase Chain Reaction Assays on Pectoralis major Muscle in Chicken (Gallus gallus )," PLOS ONE, Public Library of Science, vol. 10(5), pages 1-15, May.
    • Handle: RePEc:plo:pone00:0127935
    DOI: 10.1371/journal.pone.0127935
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    References listed on IDEAS

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    1. Daijun Ling, 2012. "SASqPCR: Robust and Rapid Analysis of RT-qPCR Data in SAS," PLOS ONE, Public Library of Science, vol. 7(1), pages 1-5, January.
    2. María Martínez-Giner & José Luis Noguera & Ingrid Balcells & Amanda Fernández-Rodríguez & Ramona N Pena, 2013. "Selection of Internal Control Genes for Real-Time Quantitative PCR in Ovary and Uterus of Sows across Pregnancy," PLOS ONE, Public Library of Science, vol. 8(6), pages 1-9, June.
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