Studying avian cytokine expression profile by Real Time PCR

JUAN MANUEL CARBALLEDA; SILVINA CHIMENO ZOTH; EVANGELINA GOMEZ; GUIDO KÖNIG; MARÍA JOSÉ GRAVISACO; ANALÍA BERINSTEIN

Viña del Mar

Congreso; IX Congreso Latinoamericano de Inmunología; 2009

Asociación Latinoamericana de Inmunología

Antibody-based techniques have been widely employed to study cytokine production in mammals. In the avian immunity field, the lack of enough commercial antibodies for cytokine analysis constitutes a limiting factor. Nevertheless, the availability of the genomic sequence of Gallus gallus has allowed the study of avian cytokine expression profiles by Real Time PCR. The aim of the present work was to optimize the conditions to establish a methodology for the quantitative analysis of cytokine expression in chickens. Fragments of 200 bp coding for different avian cytokines (IFNg, IFNa, TNFa, IL-6, IL-8, IL-15, TGFb) were amplified by RT-PCR using RNA extracted from spleen of SPF chickens. Amplification products were cloned and their sequence identity was confirmed. Recombinant DNAs were quantified and used to make standard curves for each cytokine. The same procedure was applied for the housekeeping gene (GAPDH). The efficiency and specificity of each reaction were analyzed by means of the standard curve slope and the dissociation curve, respectively. Initial mRNA concentration of each cytokine and GAPDH were calculated by extrapolation of its Ct value in the corresponding standard curve. The developed methodology constitutes a very valuable tool to be used in future studies of chicken immune response.