A simple method for 3¢-labeling of RNA

We describe a simple method for 3′-end labeling RNAs of known sequence. A short DNA template is designed to anneal to the 3′-end of the RNA, with a two nucleotide 5 ′ overhang of 3′-TA-5′, 3′-TG-5 ′ or 3′-TC-5′. The Klenow fragment of DNA polymerase I can then cleanly and efficiently extend the 3′-end of the RNA by the incorpor-ation of a single α-32P-labeled dATP residue. This method can be used to label one RNA in a mixture of RNAs, or to label 5′-blocked RNAs such as mRNA. Two methods are commonly used for 3′-end labeling RNA: T4 RNA ligase with 3′,5′[5′-32P]pCp (1,2), and poly(A) polymerase with [α-32P]cordycepin 5′-triphosphate (CoTP or 3′-deoxy-ATP) (3,4). Labeling with T4 RNA ligase requires high concentrations of pCp and enzyme, and is less efficient with long RNAs (3)