Document Detail

A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA.
MedLine Citation:
PMID:  19180082     Owner:  NLM     Status:  MEDLINE    
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.
Marc Thiry; Françoise Lamaye; Nicolas Thelen; Aurore Chatron-Colliet; Nathalie Lalun; Hélène Bobichon; Dominique Ploton
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Nature protocols     Volume:  3     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2008  
Date Detail:
Created Date:  2009-01-30     Completed Date:  2009-04-01     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  1997-2004     Citation Subset:  IM    
Laboratoire de biologie cellulaire et tissulaire, Université de Liège, Institut d'anatomie (Bât L3), 20 rue de Pitteurs, 4020 Liège, Belgique.
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MeSH Terms
Alpha-Amanitin / pharmacology
Cell Culture Techniques
Dactinomycin / pharmacology
Epithelial Cells / drug effects,  metabolism*
Hela Cells
RNA, Ribosomal / metabolism*
Transcriptional Activation
Uridine Triphosphate / analogs & derivatives
Reg. No./Substance:
0/Alpha-Amanitin; 0/RNA, Ribosomal; 3398-50-3/5-bromouridine triphosphate; 50-76-0/Dactinomycin; 63-39-8/Uridine Triphosphate

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