Source:http://linkedlifedata.com/resource/pubmed/id/11017702
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
2
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| pubmed:dateCreated |
2000-11-2
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| pubmed:abstractText |
Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human beta-globin open reading frame/c-myc 3'-untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to beta-actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serum-starved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and TaqMan) PCR methods were used to assay the identical cDNA. The real-time methods produced a 4- to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r(2) obtained from the linear regression of the first-order mRNA decay plots was SYBR green > TaqMan > probe hybridization > band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.
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| pubmed:grant | |
| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
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| pubmed:chemical | |
| pubmed:status |
MEDLINE
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| pubmed:month |
Oct
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| pubmed:issn |
0003-2697
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| pubmed:author | |
| pubmed:copyrightInfo |
Copyright 2000 Academic Press.
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| pubmed:issnType |
Print
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| pubmed:day |
15
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| pubmed:volume |
285
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| pubmed:owner |
NLM
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| pubmed:authorsComplete |
Y
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| pubmed:pagination |
194-204
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| pubmed:dateRevised |
2007-11-14
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| pubmed:meshHeading |
pubmed-meshheading:11017702-3T3 Cells,
pubmed-meshheading:11017702-Animals,
pubmed-meshheading:11017702-Cells, Cultured,
pubmed-meshheading:11017702-Computer Systems,
pubmed-meshheading:11017702-DNA Primers,
pubmed-meshheading:11017702-Fluorescent Dyes,
pubmed-meshheading:11017702-Genes, fos,
pubmed-meshheading:11017702-Genes, myc,
pubmed-meshheading:11017702-Globins,
pubmed-meshheading:11017702-Humans,
pubmed-meshheading:11017702-Linear Models,
pubmed-meshheading:11017702-Mice,
pubmed-meshheading:11017702-RNA, Messenger,
pubmed-meshheading:11017702-Reverse Transcriptase Polymerase Chain Reaction,
pubmed-meshheading:11017702-Sensitivity and Specificity,
pubmed-meshheading:11017702-Time Factors
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| pubmed:year |
2000
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| pubmed:articleTitle |
Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods.
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| pubmed:affiliation |
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, Washington 99164-6534, USA. Schmittg@mail.wsu.edu
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| pubmed:publicationType |
Journal Article,
Comparative Study,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't,
Evaluation Studies
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