Performance validation of quantitative polymerase chain reaction assays for measuring swine proinflammatory and immunomodulatory cytokine gene expression

Authors

  • Obdulio García Nicolás Department of Anatomy and Comparative Pathology, Faculty of Veterinary Medicine, Murcia University, “Mare Nostrum Excellence Campus – 3738”, 30100 Murcia, Spain
  • Juan José Quereda Spanish National Center of Biotechnology (CSIC), C/Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain
  • Jaime Gómez Laguna CICAP – Agrifood Research Centre, 14400 Pozoblanco, Córdoba, Spain
  • Irene Magdalena Rodríguez-Gómez Department of Anatomy and Comparative Pathology, Faculty of Veterinary Medicine, Cordoba University, Agrifood Campus of International Excellence - CeiA3, 14071 Cordoba, Spain
  • Librado Carrasco Department of Anatomy and Comparative Pathology, Faculty of Veterinary Medicine, Cordoba University, Agrifood Campus of International Excellence - CeiA3, 14071 Cordoba, Spain
  • Guillermo Ramis Department of Animal Production, Faculty of Veterinary Medicine, Murcia University, “Mare Nostrum Excellence Campus – 3738”, 30100 Murcia, Spain
  • Francisco José Pallarés Department of Anatomy and Comparative Pathology, Faculty of Veterinary Medicine, Murcia University, “Mare Nostrum Excellence Campus – 3738”, 30100 Murcia, Spain
Keywords: swine, cytokines, quantitative PCR, validation

Supporting Agencies

  • Grant Support: Obdulio García-Nicolás was supported by a doctoral grant from the Spanish Ministry of Economy and Competition (BES-2012-0324508). This work was supported financially by the Spanish Ministry of Economy and Competition, project number AGL2009-12438/GA.

Abstract

RT-qPCR is the method of choice for the accurate detection of low quantities of mRNA due to its highersensitivity and specificity. Most of the previously published reports about swine cytokine gene expression lack information regarding the validation of the technique, which impedes the potential implementation by new users. This study was focussed on the technical validation of already published RT-qPCR assays for swine proinflammatory (IFN-α, IFN-γ, IL-12p35, IL-12p40 and TNF-α) and immunomodulatory (IL-10 and TGF-β) cytokines, and on defining the best qPCR amplification conditions for simultaneous amplification of the selected cytokines from several porcine tissue samples. The tested RT-qPCR assays here are sensitive (Efficient close to 2, Correlation coefficient higher than 0.95 and a Limit Of Detection below 305-100 mRNA copies), robust (Coefficint of Variation and Factor of Discrimination means were lower than 5 and 3%, respectively) and highly useful for the study of immune swine responses.

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Published
16-01-2019
How to Cite
García Nicolás, O., Quereda, J. J., Gómez Laguna, J., Rodríguez-Gómez, I. M., Carrasco, L., Ramis, G., & Pallarés, F. J. (2019). Performance validation of quantitative polymerase chain reaction assays for measuring swine proinflammatory and immunomodulatory cytokine gene expression. Anales de Veterinaria de Murcia, 31, 21–35. Retrieved from https://revistas.um.es/analesvet/article/view/358701
Issue
Vol. 31 (2015)
Section
Trabajos Fin de Grado/Fin de Máster

 

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