3'-end labeling of RNA with [5'-32P]Cytidine 3',5'-bis(phosphate) and T4 RNA ligase 1.

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.