title: 3'-end labeling of RNA with [5'-32P]Cytidine 3',5'-bis(phosphate) and T4 RNA ligase 1.
abstract: This protocol is used to radiolabel the 3' ends of RNAs, either synthesized by in vitro transcription or purified from cells or tissues, by ligation of [5'-(32)P]cytidine 3',5'-bis(phosphate) (pCp). [5'-(32)P]pCp can be obtained commercially or prepared in the laboratory using polynucleotide kinase to phosphorylate cytidine-3'-monophosphate (Cp) with [γ-(32)P]ATP. "Homemade" [5'-(32)P]pCp is considerably cheaper and has a higher final concentration than that obtained from commercial sources. The labeling protocol uses T4 RNA ligase 1, which covalently joins [5'-(32)P]pCp to the free 3' hydroxyl of RNA. For best labeling, [5'-(32)P]pCp should be at least equimolar or higher to available 3'-hydroxyl ends. The reaction requires overnight incubation at low temperature. At the end of the procedure, the reaction is desalted by gel filtration to remove any unincorporated [5'-(32)P]pCp.
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Nilsen
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology; D003565:Cytidine Diphosphate / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D010761:Phosphorus Radioisotopes; D014764:Viral Proteins; C032883:cytidine 3',5'-diphosphate; D003565:Cytidine Diphosphate; D012313:RNA; D011130:RNA Ligase (ATP); C477285:bacteriophage T4 RNA ligase 1