in vitro semi-quantitative determinataion of human gamma-interferon expression by rt-pcr
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abstract
secreted cytokines of th1 (t-helper)/th2 cells play an important role in the pathogenesis of many diseases. th1 cells secrete predominantly ifn-γ and il-2 which regulate cell-mediated immunity against intracellular pathogens and tumors. in this study, expression of ifn-γ was studied using semiquantitative rt-pcr. in brief, lymphocytes of a healthy donor were stimulated with pha (1μg/106 cell/ml) in cell culture at different incubation times (0, 4, 8, 12, 24, 48 and 72 hours) to express ifn-γ. total rna was extracted and cdna synthesized. a sequence (273 bp) between two oligonucleotide primers (chosen from two different exons of the ifn-γ gene sequences) was amplified using a heat-stable dna polymerase. in semi-quantitative rt-pcr, we used a serial dilution (1/2, 1/4, …) for cdna in order to determine the titer of cdna which gives visible band in agarose gel (2%) electrophoresis. results show the highest level of ifn-γ expression was achieved after 4 hours activation with pha and it was stable at least for 22 hours. then it fell to baseline level.
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Journal title:
acta medica iranicaجلد ۴۵، شماره ۱، صفحات ۷-۱۲
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