Labeling microRNA precursors for Dicer assays

Verena Schlösser; Jonathan Hall

doi:10.1016/j.ab.2019.05.008

Labeling microRNA precursors for Dicer assays

Anal Biochem. 2019 Aug 15:579:35-37. doi: 10.1016/j.ab.2019.05.008. Epub 2019 May 12.

Authors

Verena Schlösser¹, Jonathan Hall²

Affiliations

¹ Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zürich, Vladimir-Prelog-Weg 4, CH-8093, Zürich, Switzerland.
² Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zürich, Vladimir-Prelog-Weg 4, CH-8093, Zürich, Switzerland. Electronic address: jonathan.hall@pharma.ethz.ch.

PMID: 31091428
DOI: 10.1016/j.ab.2019.05.008

Abstract

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.

Keywords: 3p strand; Dicer assay; [(32)P]-labeling; isomiR; microRNA.

MeSH terms

MicroRNAs / chemistry*
RNA Precursors / chemistry*
Ribonuclease III / chemistry*
Staining and Labeling / methods*

Substances

MicroRNAs
RNA Precursors
Ribonuclease III