تعیین نیمه کمی میزان بیان mRNA اینترفرون گاما با استفاده از روش RT-PCR

Cells communicate with one another not only by cell-cell contact, but also via the elaboration of soluble mediators or cytokines. The functionally distinct repertoire of secreted cytokines of Th1/Th2 cells has been shown to play an important role in the pathogenesis of inflammatory diseases. Effector Th1 cells secretes predominantly IFN- γ and IL-2 and regulates cell-mediated immunity against intracellular pathogens. Regulatory cytokines    are likely to be secreted in small quantities in response to specific stimuli and taken by responder cells. As a result, many scientists determine cytokine’s m-RNA expression by using a more sensitive technique, RT-PCR.           The expression of IFN- γ -mRNA was studied using semiquantitative RT- PCR. Lymphocytes were stimulated by phytohaemagglutinin (PHA) (1μg/106) in culture medium (incubation time: 0,4,8,12,24,48 and72 hour) and total RNA extracted and cDNA synthesized. IFN- γ was detected by the    RT-PCR (reverse transcription-polymerase chain reaction) and semi-quantitative-RT-PCR methods, in which sequences (273bp) between two oligonucleotide primers (chosen from two exons of the IFN- γgene sequences) were amplified using a heat-stable DNA polymerase. In semi-quantitative RT-PCR we used a serial dilution (1/2,1/4…) of cDNA in order to determine the titer of cDNA for that will give visible band in 2% agarose gel  electrophoresis.           Results showed that after 4h incubation express highest level of IFN- γ- mRNA and it is stable until 24 hour after that. Then it falls to baseline level.           In order to study the IFN- γ gene expression kinetic compare results with other studies show that RT-PCR detection of IFN- γ level is more sensitive than other methods like Elisa and for maximum expression of IFN- γ gene 4h incubation of lymphocytes with PHA is sufficient.