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

espanolLa alta sensibilidad y especificidad de la RT-qPCR le hacen el metodo de eleccion para la deteccion precisa de bajas cantidades de ARNm. La mayoria de articulos previamente publicados sobre expresion de citocinas porcinas carecen de informacion acerca de la validacion de la tecnica empleada, lo que dificulta su aplicacion por nuevos usuarios. El presente estudio se centra en la validacion de tecnicas de RT-qPCR previamente publicadas para citocinas proinflamatorias (IFN-α, IFN-γ, IL-12p35, IL-12p40 y TNF-α) e inmunomoduladoras (IL-10 y TGF-β). Ademas se definen las mejores condiciones de amplificacion simultanea para qPCR de las citadas citocinas en tejidos porcinos. Los ensayos de RT-qPCR evaluados son sensibles (Eficiencia cercana a 2, coeficiente de correlacion mayor de 0.95 y un limite de deteccion entre 305-100 copias de ARNm), son robustas (coeficiente de variacion y factor de discriminacion menores de 5 y 3%, respectivamente), y don altamente utiles para el estudio de la respuesta inmune en el cerdo EnglishRT-qPCR is the method of choice for the accurate detection of low quantities of mRNA due to its higher sensitivity 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.