Northern blotting for human pre-tRNA and tRNA-derived RNAs.

Abstract

Northern blotting (NB) is a classic method for visualizing the length as well as the amount of specific RNA using gel separation and hybridization probes. As transfer RNA-derived RNAs (tDRs) are generated from mature tRNAs or pre-tRNAs, the ratio of tDR to mature tRNA or pre-tRNA will be a useful information about the efficiency of tDR production. By designing NB probes which hybridize to a mature tRNA of interest, the blot can simultaneously visualize the amount of tDRs as well as mature tRNAs and pre-tRNAs originated from the same gene, which is a significant advantage of NB. In this chapter, we present a protocol for the detection of tDRs or pre-tRNAs by NB using denaturing polyacrylamide gel electrophoresis and Digoxigenin-dUTP-tailed oligo DNA probes. Through example experiments, we show that tDRs originating from the same mature tRNA can be differentiated based on their length. We also show that our method can be applied to the evaluation of pre-tRNA processing.