Volume 10, Issue 2 (Scientific Journal of Hamadan University of Medical Sciences-Summer 2003)                   Avicenna J Clin Med 2003, 10(2): 5-11 | Back to browse issues page

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Tavakol Afshari J, Zamani A, Behravan J, Shisheeyan B, Saifi B. In Vitro Semi-Quantitative Determination of Human Gamma-Interferon-mRNA Level by RT-PCR. Avicenna J Clin Med. 2003; 10 (2) :5-11
URL: http://sjh.umsha.ac.ir/article-1-664-en.html
Abstract:   (1648 Views)

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.

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Type of Study: Original | Subject: Special

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