Paper Information

Journal:   AVICENNA JOURNAL OF CLINICAL MEDICINE (SCIENTIFIC JOURNAL OF HAMADAN UNIVERSITY OF MEDICAL SCIENCES AND HEALTH SERVICES)   Summer 2003 , Volume 10 , Number 2; Page(s) 5 To 11.
 
Paper: 

IN VITRO SEMI-QUANTITATIVE DETERMINATION OF HUMAN GAMMA-INTERFERON-MRNA LEVEL BY RT-PCR

 
 
Author(s):  AFSHARI J.T., ZAMANI ALI REZA*, BEHRAVAN J., SHISHEEYAN B., SAIFI B.
 
* DEPARTMENT OF IMMUNOLOGY, SCHOOL OF MEDICINE, HAMADAN UNIVERSITY OF MEDICAL SCIENCES & HEALTH SERVICES, HAMADAN, IRAN
 
Abstract: 

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- γ gene 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.

 
Keyword(s): IFN- Y, PHA ACTIVATED LYMPHOCYTES, SEMI-QUANTITATIVE-RT-PCR, REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION
 
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