A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA.

Alpha-Amanitin/pharmacology; Cell Culture Techniques; Dactinomycin/pharmacology; Epithelial Cells/drug effects/metabolism; Hela Cells; Humans; Kinetics; RNA, Ribosomal/metabolism; Transcriptional Activation; Uridine Triphosphate/analogs & derivatives

Abstract :

[en] 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.

Disciplines :

Anatomy (cytology, histology, embryology...) & physiology

Author, co-author :

Thiry, Marc ; Université de Liège - ULiège > Département des sciences de la vie > Biologie cellulaire

Lamaye, Françoise ; Université de Liège - ULiège > Département des sciences de la vie > Biologie cellulaire

Thelen, Nicolas ; Université de Liège - ULiège > Département des sciences de la vie > Biologie cellulaire

Chatron-Colliet, Aurore

Lalun, Nathalie

Bobichon, Helene

Ploton, Dominique

Language :

English

Title :

A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA.

Publication date :

2008

Journal title :

Nature Protocols

ISSN :

1754-2189

eISSN :

1750-2799

Publisher :

Nature Publishing Group, United Kingdom

Volume :

Issue :

Pages :

1997-2004

Peer reviewed :

Peer Reviewed verified by ORBi

Available on ORBi :

since 28 November 2009

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