A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA.
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.
Thiry
Marc
M
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
Lamaye
Françoise
F
Thelen
Nicolas
N
Chatron-Colliet
Aurore
A
Lalun
Nathalie
N
Bobichon
Hélène
H
Ploton
Dominique
D
D053959:Alpha-Amanitin / Q000494:pharmacology; D018929:Cell Culture Techniques; D003609:Dactinomycin / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D012335:RNA, Ribosomal / Q000378:metabolism*; D015533:Transcriptional Activation; D014544:Uridine Triphosphate / Q000031:analogs & derivatives
D053959:Alpha-Amanitin; D012335:RNA, Ribosomal; D003609:Dactinomycin; C018009:5-bromouridine triphosphate; D014544:Uridine Triphosphate