Labeling microRNA precursors for Dicer assays

Excision of the terminal loop of precursor microRNAs (pre-miRNA) by Dicer generates miRNA duplexes, comprising 5p and 3p strands. Dicer often cleaves the RNA at more than one site, producing mature miRNAs with heterogeneous 3' and 5' ends. As pre-miRNAs are most conveniently labeled at their 5' ends, standard in vitro Dicer assays usually assay only 5p miRNA strands. In this work we present a straightforward protocol using the same [³²P] isotope for both strands by placing an internal label into the 3p strand, thereby providing a tool to investigate miRNA processing, strand selection and isomiR formation for any pre-miRNA.