IN VITRO SEMI-QUANTITATIVE DETERMINATAION OF HUMAN GAMMA-INTERFERON EXPRESSION BY RT-PCR

Cytokines secreted by th1 (t-helper)/th2 cells play an important role in the pathogenesisof many diseases. th1 cells secrete predominantly ifn-م and il-2 which regulate cell-mediatedimmunity against intracellular pathogens and tumors. in this study, expression of ifn-م was studiedusing semiquantitative real time polymerase chain reaction (rt-pcr). in brief, lymphocytes of ahealthy donor were stimulated with pha (1ىg/106 cell/ml) in cell culture at different incubation times(0, 4, 8, 12, 24, 48 and 72 hours) to express ifn-م. total rna was extracted and cdna synthesized. asequence (273 bp) between two oligonucleotide primers (chosen from two different exons of the ifn-مgene sequences) was amplified using a heat-stable dna polymerase. in semi-quantitative rt-pcr, weused a serial dilution for cdna in order to determine the titer of cdna which gives visible band inagarose gel (2%) electrophoresis. results showed that the highest level of ifn-م expression wasachieved after 4 hours activation with pha and it was stable at least for 22 hours. then it fell tobaseline level. cytokine detection using rt-pcr will provide useful clinical information.