| A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA. | |
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MedLine Citation:
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PMID: 19180082 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5'-triphosphate into RNA of living cells by lipofection followed by immunocytological detection of BrRNAs. The use of the same antibody identified either with fluorescence or with gold particles revealed the three-dimensional organization of sites containing labeled RNAs or their precise localization by using confocal and ultrastructural microscopy, respectively. Comparison of three-dimensional reconstruction obtained from the series of optical sections and ultrathin sections was extremely fruitful to describe topological and spatial dynamics of RNAs from their synthesis site inside the nucleus to the cytoplasm. Combined with immunolocalization of proteins involved in different nuclear activities and with highly resolved three-dimensional visualizations of the labelings, this method should also provide a significant contribution to our understanding of the functional, volumic organization of the cell nucleus. The entire protocol can be completed in approximately 10 d. |
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Authors:
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Marc Thiry; Françoise Lamaye; Nicolas Thelen; Aurore Chatron-Colliet; Nathalie Lalun; Hélène Bobichon; Dominique Ploton |
Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Nature protocols Volume: 3 ISSN: 1750-2799 ISO Abbreviation: Nat Protoc Publication Date: 2008 |
Date Detail:
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Created Date: 2009-01-30 Completed Date: 2009-04-01 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 101284307 Medline TA: Nat Protoc Country: England |
Other Details:
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Languages: eng Pagination: 1997-2004 Citation Subset: IM |
Affiliation:
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Laboratoire de biologie cellulaire et tissulaire, Université de Liège, Institut d'anatomie (Bât L3), 20 rue de Pitteurs, 4020 Liège, Belgique. mthiry@ulg.ac.be |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Alpha-Amanitin
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pharmacology Cell Culture Techniques Dactinomycin / pharmacology Epithelial Cells / drug effects, metabolism* Hela Cells Humans Kinetics RNA, Ribosomal / metabolism* Transcriptional Activation Uridine Triphosphate / analogs & derivatives |
| Chemical | |
Reg. No./Substance:
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0/Alpha-Amanitin; 0/RNA, Ribosomal; 3398-50-3/5-bromouridine triphosphate; 50-76-0/Dactinomycin; 63-39-8/Uridine Triphosphate |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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