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

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AFSHARI J.T.; ZAMANI ALI REZA; BEHRAVAN J.; SHISHEEYAN B.; SAIFI B.

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Journal: Avicenna Journal of Clinical Medicine Year:2003 | Volume:10 | Issue:2 Page(s): 5-11

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Title

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

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Keywords

IFN- YQ2

PHA ACTIVATED LYMPHOCYTESQ4

SEMI-QUANTITATIVE-RT-PCRQ2

REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTIONQ2

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.

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APA: Copy

AFSHARI, J.T., ZAMANI, ALI REZA, BEHRAVAN, J., SHISHEEYAN, B., & SAIFI, B.. (2003). IN VITRO SEMI-QUANTITATIVE DETERMINATION OF HUMAN GAMMA-INTERFERON-MRNA LEVEL BY RT-PCR. AVICENNA JOURNAL OF CLINICAL MEDICINE (SCIENTIFIC JOURNAL OF HAMADAN UNIVERSITY OF MEDICAL SCIENCES AND HEALTH SERVICES), 10(2), 5-11. SID. https://sid.ir/paper/17322/en

Vancouver: Copy

AFSHARI J.T., ZAMANI ALI REZA, BEHRAVAN J., SHISHEEYAN B., SAIFI B.. IN VITRO SEMI-QUANTITATIVE DETERMINATION OF HUMAN GAMMA-INTERFERON-MRNA LEVEL BY RT-PCR. AVICENNA JOURNAL OF CLINICAL MEDICINE (SCIENTIFIC JOURNAL OF HAMADAN UNIVERSITY OF MEDICAL SCIENCES AND HEALTH SERVICES)[Internet]. 2003;10(2):5-11. Available from: https://sid.ir/paper/17322/en

IEEE: Copy

J.T. AFSHARI, ALI REZA ZAMANI, J. BEHRAVAN, B. SHISHEEYAN, and B. SAIFI, “IN VITRO SEMI-QUANTITATIVE DETERMINATION OF HUMAN GAMMA-INTERFERON-MRNA LEVEL BY RT-PCR,” AVICENNA JOURNAL OF CLINICAL MEDICINE (SCIENTIFIC JOURNAL OF HAMADAN UNIVERSITY OF MEDICAL SCIENCES AND HEALTH SERVICES), vol. 10, no. 2, pp. 5–11, 2003, [Online]. Available: https://sid.ir/paper/17322/en

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