In Vitro Semi-Quantitative Determination of Human Gamma-Interferon-mRNA Level by RT-PCR
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. Effecto...
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| Format: | Article |
| Language: | fas |
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Hamadan University of Medical Sciences
2003-09-01
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| Series: | پزشکی بالینی ابن سینا |
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| Online Access: | http://sjh.umsha.ac.ir/article-1-664-en.html |
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| author | Jalil Tavakol Afshari Alireza Zamani Javad Behravan Behrouz Shisheeyan Bita Saifi |
| author_facet | Jalil Tavakol Afshari Alireza Zamani Javad Behravan Behrouz Shisheeyan Bita Saifi |
| author_sort | Jalil Tavakol Afshari |
| collection | DOAJ |
| description | 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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| institution | Directory Open Access Journal |
| issn | 2588-722X 2588-7238 |
| language | fas |
| last_indexed | 2024-12-12T16:37:59Z |
| publishDate | 2003-09-01 |
| publisher | Hamadan University of Medical Sciences |
| record_format | Article |
| series | پزشکی بالینی ابن سینا |
| spelling | doaj.art-cf8583644bc540828531cb1a7b1a4ae92022-12-22T00:18:38ZfasHamadan University of Medical Sciencesپزشکی بالینی ابن سینا2588-722X2588-72382003-09-01102511In Vitro Semi-Quantitative Determination of Human Gamma-Interferon-mRNA Level by RT-PCRJalil Tavakol Afshari0Alireza Zamani1Javad Behravan2Behrouz Shisheeyan3Bita Saifi4 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.http://sjh.umsha.ac.ir/article-1-664-en.htmlifn- ?pha activated lymphocytessemi-quantitative-rt-pcrreverse transcriptase polymerase chain reaction |
| spellingShingle | Jalil Tavakol Afshari Alireza Zamani Javad Behravan Behrouz Shisheeyan Bita Saifi In Vitro Semi-Quantitative Determination of Human Gamma-Interferon-mRNA Level by RT-PCR پزشکی بالینی ابن سینا ifn- ? pha activated lymphocytes semi-quantitative-rt-pcr reverse transcriptase polymerase chain reaction |
| title | In Vitro Semi-Quantitative Determination of Human Gamma-Interferon-mRNA Level by RT-PCR |
| title_full | In Vitro Semi-Quantitative Determination of Human Gamma-Interferon-mRNA Level by RT-PCR |
| title_fullStr | In Vitro Semi-Quantitative Determination of Human Gamma-Interferon-mRNA Level by RT-PCR |
| title_full_unstemmed | In Vitro Semi-Quantitative Determination of Human Gamma-Interferon-mRNA Level by RT-PCR |
| title_short | In Vitro Semi-Quantitative Determination of Human Gamma-Interferon-mRNA Level by RT-PCR |
| title_sort | in vitro semi quantitative determination of human gamma interferon mrna level by rt pcr |
| topic | ifn- ? pha activated lymphocytes semi-quantitative-rt-pcr reverse transcriptase polymerase chain reaction |
| url | http://sjh.umsha.ac.ir/article-1-664-en.html |
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