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| Format: | Artículo científico |
| Language: | en |
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Instituto Nacional de Salud
2006
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| Online Access: | https://www.redalyc.org/articulo.oa?id=84309929 |
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| _version_ | 1866814511248310272 |
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| author | Omar A. Saldarriaga |
| author_facet | Omar A. Saldarriaga |
| contents | Quantification of canine cytokines using real time reverse transcriptase polymerase chain reaction Omar A. Saldarriaga Peter C. Melby Bruno L. Travi Medicina dogs cytokines Lymphocytes visceral leishmaniasis reverse transcriptase polymerase chain reaction Introduction. Canines are the principal domestic reservoirs of visceral leishmaniasis in boththe Old and New World. The development of highly sensitive and quantitative methods, such asreal time reverse transcriptase polymerase chain reaction for measurement of canine cytokineshas not been exploited in studies of visceral leishmaniasis.Objective. To standardize the relative quantification of canine IFN-g, IL-4, IL-10, IL-12p40 andIL-12p35 using real time reverse transcriptase polymerase chain reaction.Materials and methods. RNA was isolated from PBMCs from 1 yearold foxhounds and culturedwith or without Con A, LPS or Staphylococcus aureus extract. This RNA was used in one-stepreal time reverse transcriptase polymerase chain reaction to optimize the concentrations of thecytokine primers and probes, generate standard curves for each cytokine, confirm equivalentamplification efficiency of cytokine and normalizer (18S rRNA) RNA, and to quantify theexpression of the cytokine RNA. The comparative Ct method was used to determine the relativelevels of gene expression in the samples, expressed as the fold-increase relative to the controlsamples.Results. The regression coefficient for the standard curves and the amplification efficiencies ofthe cytokine and normalizer RNA indicated that the quantification was reliable over a broadconcentration range of input RNA. Relative to control cells, activation of PBMCs led to increasedexpression of IFN-g (132-fold), IL-4 (8.8-fold), IL-10 (7.2-fold), and IL-12p40 (275-fold). Basalexpression of IL-12p35 was also detected.Conclusion. This approach provides several advantages over conventional assays for cytokinemeasurement and can be exploited in the study of the immunopathogenesis and immunity incanine leishmaniasis. 2006 artículo científico 0120-4157 https://www.redalyc.org/articulo.oa?id=84309929 en http://www.redalyc.org/revista.oa?id=843 Biomédica application/pdf Instituto Nacional de Salud Biomédica (Colombia) Num.1 Vol.26 |
| format | Artículo científico |
| id | redalyc_84309929 |
| language | en |
| publishDate | 2006 |
| publisher | Instituto Nacional de Salud |
| spellingShingle | Quantification of canine cytokines using real time reverse transcriptase polymerase chain reaction Omar A. Saldarriaga Medicina dogs cytokines Lymphocytes visceral leishmaniasis reverse transcriptase polymerase chain reaction Quantification of canine cytokines using real time reverse transcriptase polymerase chain reaction Omar A. Saldarriaga Peter C. Melby Bruno L. Travi Medicina dogs cytokines Lymphocytes visceral leishmaniasis reverse transcriptase polymerase chain reaction Introduction. Canines are the principal domestic reservoirs of visceral leishmaniasis in boththe Old and New World. The development of highly sensitive and quantitative methods, such asreal time reverse transcriptase polymerase chain reaction for measurement of canine cytokineshas not been exploited in studies of visceral leishmaniasis.Objective. To standardize the relative quantification of canine IFN-g, IL-4, IL-10, IL-12p40 andIL-12p35 using real time reverse transcriptase polymerase chain reaction.Materials and methods. RNA was isolated from PBMCs from 1 yearold foxhounds and culturedwith or without Con A, LPS or Staphylococcus aureus extract. This RNA was used in one-stepreal time reverse transcriptase polymerase chain reaction to optimize the concentrations of thecytokine primers and probes, generate standard curves for each cytokine, confirm equivalentamplification efficiency of cytokine and normalizer (18S rRNA) RNA, and to quantify theexpression of the cytokine RNA. The comparative Ct method was used to determine the relativelevels of gene expression in the samples, expressed as the fold-increase relative to the controlsamples.Results. The regression coefficient for the standard curves and the amplification efficiencies ofthe cytokine and normalizer RNA indicated that the quantification was reliable over a broadconcentration range of input RNA. Relative to control cells, activation of PBMCs led to increasedexpression of IFN-g (132-fold), IL-4 (8.8-fold), IL-10 (7.2-fold), and IL-12p40 (275-fold). Basalexpression of IL-12p35 was also detected.Conclusion. This approach provides several advantages over conventional assays for cytokinemeasurement and can be exploited in the study of the immunopathogenesis and immunity incanine leishmaniasis. 2006 artículo científico 0120-4157 https://www.redalyc.org/articulo.oa?id=84309929 en http://www.redalyc.org/revista.oa?id=843 Biomédica application/pdf Instituto Nacional de Salud Biomédica (Colombia) Num.1 Vol.26 |
| title | Quantification of canine cytokines using real time reverse transcriptase polymerase chain reaction |
| topic | Medicina dogs cytokines Lymphocytes visceral leishmaniasis reverse transcriptase polymerase chain reaction |
| url | https://www.redalyc.org/articulo.oa?id=84309929 |