| Rapid detection of apoptosis through real-time reverse transcriptase polymerase chain reaction measurement of the small cytoplasmic RNA Y1. | |
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MedLine Citation:
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PMID: 12814625 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Apoptosis could be measured in mammalian cells by measuring the degradation of the small cytoplasmic human RNA Y1 (hY1) by real-time quantitative fluorescent reverse transcriptase polymerase chain reaction (RT-PCR). In FAS-antibody-treated Jurkat T cell leukemia cells degradation of hY1 occurred rapidly and was complete at about 6h. As in apoptotic Jurkat cells, protein synthesis is arrested only after about 12h; this implies that protein synthesis can occur without scRNA-Y1. The degradation of hY1 could be blocked with peptide-based inhibitors of caspase 8 and with lower efficacy with caspases 1 and 3 and with ZnSO4. No effects were observed after inhibition of caspases 2, 6, and 9. Degradation of hY1 could also be demonstrated after treatment of A549 lung carcinoma cells treated with Staurosporin, Paclitaxel, or the histone deacetylase inhibitor LAQ824. RT-PCR systems based on SYBR Green, Amplifluor Uniprimer, or 5' nuclease Taqman could be used with increasing sensitivity. This apoptosis assay requires quantities of total cell RNA equivalent to only a few tissue culture cells and is especially suited to measure apoptosis in projects where RNA samples are already available from gene expression studies. |
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Authors:
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Fred A M Asselbergs; Roland Widmer |
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Publication Detail:
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Type: Comparative Study; Journal Article |
Journal Detail:
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Title: Analytical biochemistry Volume: 318 ISSN: 0003-2697 ISO Abbreviation: Anal. Biochem. Publication Date: 2003 Jul |
Date Detail:
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Created Date: 2003-06-19 Completed Date: 2004-02-20 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 0370535 Medline TA: Anal Biochem Country: United States |
Other Details:
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Languages: eng Pagination: 221-9 Citation Subset: IM |
Affiliation:
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Department of Functional Genomics, Novartis Pharma AG, WSJ-360.601, CH-4002 Basel, Switzerland. fred_am.asselbergs@pharma.novartis.com |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Antigens, CD95
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metabolism Apoptosis / genetics* Biological Markers / analysis Caspases / antagonists & inhibitors, metabolism Cell Line, Tumor Enzyme Inhibitors / pharmacology Humans Jurkat Cells Kinetics RNA Stability RNA, Small Cytoplasmic / analysis*, metabolism Reproducibility of Results Reverse Transcriptase Polymerase Chain Reaction / methods* Sensitivity and Specificity |
| Chemical | |
Reg. No./Substance:
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0/Antigens, CD95; 0/Biological Markers; 0/Enzyme Inhibitors; 0/RNA, Small Cytoplasmic; EC 3.4.22.-/Caspases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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