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′-32P]cytidine 3′,5′-bis(phosphate) (pCp). [5′-32P]pCp can be obtained commercially or prepared in the laboratory using polynucleotide kinase to phosphorylate cytidine-3′-monophosphate (Cp) with [γ-32P]ATP. “Homemade” [5′-32P]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′-32P]pCp to the free 3′ hydroxyl of RNA. For best labeling, [5′-32P]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′-32P]pCp.