rdf:type |
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lifeskim:mentions |
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pubmed:issue |
5
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pubmed:dateCreated |
2003-5-19
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pubmed:abstractText |
Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Actins,
http://linkedlifedata.com/resource/pubmed/chemical/Cytokines,
http://linkedlifedata.com/resource/pubmed/chemical/Fluorescent Dyes,
http://linkedlifedata.com/resource/pubmed/chemical/Indicators and Reagents,
http://linkedlifedata.com/resource/pubmed/chemical/Interferon-gamma,
http://linkedlifedata.com/resource/pubmed/chemical/Interleukin-12,
http://linkedlifedata.com/resource/pubmed/chemical/Interleukin-12 Subunit p40,
http://linkedlifedata.com/resource/pubmed/chemical/Interleukin-2,
http://linkedlifedata.com/resource/pubmed/chemical/Ionomycin,
http://linkedlifedata.com/resource/pubmed/chemical/Organic Chemicals,
http://linkedlifedata.com/resource/pubmed/chemical/Protein Subunits,
http://linkedlifedata.com/resource/pubmed/chemical/RNA, Messenger,
http://linkedlifedata.com/resource/pubmed/chemical/SYBR Green I,
http://linkedlifedata.com/resource/pubmed/chemical/Tetradecanoylphorbol Acetate,
http://linkedlifedata.com/resource/pubmed/chemical/Transforming Growth Factor beta
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pubmed:status |
MEDLINE
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pubmed:month |
May
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pubmed:issn |
0300-9475
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
57
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
439-45
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pubmed:dateRevised |
2008-11-21
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pubmed:meshHeading |
pubmed-meshheading:12753500-Actins,
pubmed-meshheading:12753500-Animals,
pubmed-meshheading:12753500-Computer Systems,
pubmed-meshheading:12753500-Cost-Benefit Analysis,
pubmed-meshheading:12753500-Costs and Cost Analysis,
pubmed-meshheading:12753500-Cytokines,
pubmed-meshheading:12753500-Female,
pubmed-meshheading:12753500-Fluorescent Dyes,
pubmed-meshheading:12753500-Gene Expression Profiling,
pubmed-meshheading:12753500-Indicators and Reagents,
pubmed-meshheading:12753500-Interferon-gamma,
pubmed-meshheading:12753500-Interleukin-12,
pubmed-meshheading:12753500-Interleukin-12 Subunit p40,
pubmed-meshheading:12753500-Interleukin-2,
pubmed-meshheading:12753500-Ionomycin,
pubmed-meshheading:12753500-Lymphocyte Activation,
pubmed-meshheading:12753500-Mice,
pubmed-meshheading:12753500-Mice, Inbred BALB C,
pubmed-meshheading:12753500-Oligonucleotide Array Sequence Analysis,
pubmed-meshheading:12753500-Organic Chemicals,
pubmed-meshheading:12753500-Polymerase Chain Reaction,
pubmed-meshheading:12753500-Protein Subunits,
pubmed-meshheading:12753500-RNA, Messenger,
pubmed-meshheading:12753500-Sensitivity and Specificity,
pubmed-meshheading:12753500-Spleen,
pubmed-meshheading:12753500-T-Lymphocytes,
pubmed-meshheading:12753500-Tetradecanoylphorbol Acetate,
pubmed-meshheading:12753500-Transforming Growth Factor beta
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pubmed:year |
2003
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pubmed:articleTitle |
Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I.
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pubmed:affiliation |
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D F, México.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't,
Evaluation Studies
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