3′-End Labeling of RNA with Yeast Poly(A) Polymerase and 3′-Deoxyadenosine 5′-[α-³²P]Triphosphate

Abstract

This protocol is used to label RNA molecules (in vitro–synthesized or in vivo–purified RNA molecules) that have free 3′-hydroxyl termini. The reaction is performed in 10 min using yeast poly(A) polymerase and 3′-deoxyadenosine 5′-[α-³²P]triphosphate (cordycepin 5′-[α-³²P]triphosphate), a chain-terminating nucleotide. At the end of the procedure, the reaction is desalted by gel filtration to remove any unincorporated nucleotides.