Monitoring of Chicken RNA Integrity as a Function of Prolonged Postmortem Duration

  • Malila, Yuwares (National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency) ;
  • Srimarut, Yanee (National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency) ;
  • U-chupaj, Juthawut (Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University) ;
  • Strasburg, Gale (Department of Food Science and Human Nutrition, Michigan State University) ;
  • Visessanguan, Wonnop (National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency)
  • Received : 2015.02.26
  • Accepted : 2015.05.04
  • Published : 2015.11.01


Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major) and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR) as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6) immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem). The delayed postmortem duration did not significantly affect $A_{260}/A_{280}$ and $A_{260}/A_{230}$ ($p{\geq}0.05$), suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05), the value was not statistically different between 20 min and 6 h samples ($p{\geq}0.05$), indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), peptidylprolylisomerase A (PPIA) and TATA box-binding protein (TBP) as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and peroxisome proliferator-activated receptor delta (PPARD) were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05). Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05). The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken skeletal muscle. For the best RNA quality, chicken skeletal muscle should be immediately collected after evisceration or within 20 min postmortem, and rapidly preserved by deep freezing.


Gallus gallus;Skeletal Muscle;Postmortem Duration;RNA Integrity;Gene Expression


Supported by : National Center for Genetic Engineering and Biotechnology


  1. Bahar, B., F. J. Monahan, A. P. Moloney, O. Schmidt, D. E. MacHugh, and T. Sweeney. 2007. Long-term stability of RNA in post-mortem bovine skeletal muscle, liver and subcutaneous adipose tissues. BMC Mol. Biol. 29:108.
  2. Bahar, B., J. V. O'Doherty, and T. Sweeney. 2012. Assessment of RNA integrity in the postmortem pig colonic tissue ex vivo. J. Anim. Sci. 90 Suppl 4:22-24.
  3. Bustin, S. A. and T. Nolan. 2004. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J. Biomol. Tech. 15:155-166.
  4. Cirera, S. 2013. Highly efficient method for isolation of total RNA from adipose tissue. BMC Res. Notes. 6:472.
  5. de Fremery, D. and M. F. Pool. 1960. Biochemistry of chicken muscle as related to rigor mortis and tenderization. J. Food Sci. 25:73-87.
  6. Eadmusik, S., C. Molette, X. Fernandez, and H. Remignon. 2011Are one early muscle pH and one early temperature measurement sufficient to detect PSE breast meat in turkeys? Br. Poult. Sci. 52:177-188.
  7. Fleige, S. and M. W. Pfaffl. 2006. RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Aspects Med. 27:126-139.
  8. Fontanesi, L., M. Colombo, F. Beretti, and V. Russo. 2008. Evaluation of post mortem stability of porcine skeletal muscle RNA. Meat Sci. 80:1345-1351.
  9. Garcia, R. G., L. W. de Freitas, A. W. Schwingel, R. M. Farias, F. R. Caldara, A. M. A. Gabriel, J. D. Graciano, C. M. Komiyama, and I. C. L. Almedia Paz. 2010. Incidence and physical properties of PSE chicken meat in a commercial processing plant. Rev. Bras. Cienc. Avic. 12:233-237.
  10. Imbeaud, S., E. Graudens, V. Boulanger, X. Barlet, P. Zaborski, E. Eveno, O. Mueller, A. Schroeder, and C. Auffray. 2005. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucl. Acids Res. 33:e56.
  11. Koppelkamm, A., B. Vennemann, S. Lutz-Bonengel, T. Fracasso, and M. Vennemann. 2011. RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays. Int. J. Legal Med. 125:573-580.
  12. Le Bihan-Duval, E. 2004. Genetic variability within and between breeds of poultry technological meat quality. Worlds Poult. Sci. J. 60:331-340.
  13. Livak, K. J. and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the $2^{{-}{\Delta}{\Delta}CT}$ Method. Methods 25:402-408.
  14. Malila, Y., R. J. Tempelman, K. R. Sporer, C. W. Ernst, S. G. Velleman, K. M. Reed, and G. M. Strasburg. 2013. Differential gene expression between normal and pale, soft, and exudative turkey meat. Poult. Sci. 92:1621-1633.
  15. Mullen, A. M., P. C. Stapleton, D. Corcoran, R. M. Hamill, and A. White. 2006. Understanding meat quality through the application of genomic and proteomic approaches. Meat Sci. 74:3-16.
  16. Rasband, W. S. 2012. ImageJ. U. S. National Institutes of Health. Accessed September 9, 2014.
  17. Sambrook, J., E. F. Fristsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, New York, NY, USA.
  18. Sanoudou, D., P. B. Kang, J. N. Haslett, M. Han, L. M. Kunkel, and A. H. Beggs. 2004. Transcriptional profile of postmortem skeletal muscle. Physiol. Genomics 16:222-228.
  19. Seear, P. J. and G. E. Sweeney. 2008. Stability of RNA isolated from post-mortem tissues of Atlantic salmon (Salmo salar L.). Fish Physiol. Biochem. 34:19-24.
  20. Woelfel, R. L., C. M. Owens, E. M. Hirschler, R. Martinez-Dawson, and A. R. Sams. 2002. The characterization and incidence of pale, soft, and exudative broiler meat in a commercial processing plant. Poult. Sci. 81:579-584.