Absolute quantification of murine interleukine-۴, interleukine- ۱۰ 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 the evaluation of clinical responses to new vaccination approaches. An absolute quantification is based on a calibration curve and production of standard controls to achieve more reliable results than in relative system. In this study we attempted to construct standard controls to evaluate the murine immune response. The number of ۱۰ Balb/c mice immunized with hydatid cyst fluid subcutaneously with two week intervals to induce transcription of Th۱ and Th۲ related cytokines. Three Pairs of primers were designed to amplification of interleukine-۴, interleukine-۱۰ and interferon-γ by Oligo software. Partial sequences of three cytokine genes were cloned into pTZ۵۷T vector. Three recombinant plasmids were purified and serial dilutions were prepared. Real time qPCR carried out using SYBR-Green I fluorescence dye and standard curves were provided by the ۷۵۰۰ ABI SDS software based on the exact concentration of dilutions and the amplification plots. Results showed that this method was able to evaluate the cytokines mRNA levels less than ۰.۰۱pg (~۱۵۰ copy). We concluded that absolute real-time qPCR can be successfully applied to the quantification of antigen-induced cytokines.

Keywords:

real time PCR , Cytokine , IL۴ , IL۱۰ , IFN , γ