Labeling microRNA precursors for Dicer assays.


Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zürich, Vladimir-Prelog-Weg 4, CH-8093, Zürich, Switzerland. Electronic address: [Email]


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.


3p strand,Dicer assay,[(32)P]-labeling,isomiR,microRNA,