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

The study of cytokines gene expression is quite important in various conditions of health and disease for theevaluation of clinical responses to new vaccination approaches. an absolute quantification is based on acalibration curve and production of standard controls to achieve more reliable results than in relativesystem. in this study we attempted to construct standard controls to evaluate the murine immune response.the number of 10 balb/c mice immunized with hydatid cyst fluid subcutaneously with two week intervalsto induce transcription of th1 and th2 related cytokines. three pairs of primers were designed toamplification of interleukine-4, interleukine-10 and interferon-? by oligo software. partial sequences ofthree cytokine genes were cloned into ptz57t vector. three recombinant plasmids were purified and serialdilutions were prepared. real time qpcr carried out using sybr-green i fluorescence dye and standardcurves were provided by the 7500 abi sds software based on the exact concentration of dilutions and theamplification plots. results showed that this method was able to evaluate the cytokines mrna levels lessthan 0.01pg (~150 copy). we concluded that absolute real-time qpcr can be successfully applied to thequantification of antigen-induced cytokines.