Exploring RNA Transcription and Turnover in vivo by Using Click Chemistry

We describe a chemical method to detect RNA synthesis in cells, based on the biosynthetic incorporation of the uridine analog 5-ethynyluridine (EU) into newly transcribed RNA, on average once every 35 uridine residues in total RNA. EU-labeled cellular RNA is detected quickly and with high sensitivit...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2008-10, Vol.105 (41), p.15779-15784
Main Authors: Jao, Cindy Y., Salic, Adrian
Format: Article
Language:English
Subjects:
3T3 cells
Animal Structures - metabolism
Animals
Azides
Biological Sciences
Catalysis
Cell culture
Cells
Cells, Cultured
Clinical Laboratory Techniques
Copper
Cultured cells
DNA
Endothelial cells
Fluorescent Dyes
Hepatocytes
Kinetics
Messenger RNA
Microscopy
Nucleosides
Protein synthesis
Ribonucleic acid
RNA
RNA - analysis
RNA - biosynthesis
Small intestine
Transcription, Genetic
Uracil - analogs & derivatives
Uridine - analogs & derivatives
Uridine - metabolism
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Summary:We describe a chemical method to detect RNA synthesis in cells, based on the biosynthetic incorporation of the uridine analog 5-ethynyluridine (EU) into newly transcribed RNA, on average once every 35 uridine residues in total RNA. EU-labeled cellular RNA is detected quickly and with high sensitivity by using a copper (I)-catalyzed cycloaddition reaction (often referred to as "click" chemistry) with fluorescent azides, followed by microscopic imaging. We demonstrate the use of this method in cultured cells, in which we examine the turnover of bulk RNA after EU pulses of varying lengths. We also use EU to assay transcription rates of various tissues in whole animals, both on sections and by whole-mount staining. We find that total transcription rates vary greatly among different tissues and among different cell types within organs.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0808480105