Absolute quantification of murine interleukine-4, interleukine- 10 and interferon-γ gene transcripts using Real Time PCR

Abstract

The study of cytokines gene expression is quite important in various conditions of health and disease for the <br />evaluation of clinical responses to new vaccination approaches. An absolute quantification is based on a <br />calibration curve and production of standard controls to achieve more reliable results than in relative <br />system. In this study we attempted to construct standard controls to evaluate the murine immune response. <br />The number of 10 Balb/c mice immunized with hydatid cyst fluid subcutaneously with two week intervals <br />to induce transcription of Th1 and Th2 related cytokines. Three Pairs of primers were designed to <br />amplification of interleukine-4, interleukine-10 and interferon-γ by Oligo software. Partial sequences of <br />three cytokine genes were cloned into pTZ57T vector. Three recombinant plasmids were purified and serial <br />dilutions were prepared. Real time qPCR carried out using SYBR-Green I fluorescence dye and standard <br />curves were provided by the 7500 ABI SDS software based on the exact concentration of dilutions and the <br />amplification plots. Results showed that this method was able to evaluate the cytokines mRNA levels less <br />than 0.01pg (~150 copy). We concluded that absolute real-time qPCR can be successfully applied to the <br />quantification of antigen-induced cytokines.