Validation of miRNA-mRNA interactions by electrophoretic mobility shift assays

BMC Res Notes. 2013 Nov 12:6:454. doi: 10.1186/1756-0500-6-454.

Abstract

Background: MicroRNAs are small non-coding RNAs involved in gene expression regulation by targeting specific regions in the 3'-UTR of the mRNA of their target genes. This binding leads to a decrease in the protein levels of such genes either by mRNA degradation or mRNA destabilization and translation inhibition. The interaction between a miRNA and its target mRNAs is usually studied by co-transfection of a reporter expression vector containing the 3'-UTR region of the mRNA and an inhibitory or precursor molecule for the miRNA. This approach, however, does not measure the direct and physical interaction between a miRNA and a specific mRNA.

Findings: RNA molecules corresponding to miR-224 and to the 3'-UTR of SLC4A4 were incubated together and their interaction studied under different binding conditions using electrophoretic mobility shift assays. A direct and specific interaction between miR-224 and SLC4A4 mRNA was observed. This interaction was abolished in the presence of competitors.

Conclusions: In this study, we explored a new application for the electrophoretic mobility shift assay and we demonstrated that it is a useful alternative method to assess, in a direct and specific manner, whether a miRNA binds to a specific predicted target mRNA.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • 3' Untranslated Regions / genetics*
  • Binding Sites
  • Electrophoretic Mobility Shift Assay*
  • Gene Expression Regulation
  • Humans
  • MicroRNAs / chemistry*
  • MicroRNAs / genetics
  • Phosphorus Radioisotopes
  • RNA Stability
  • Ribonucleotides / chemistry*
  • Ribonucleotides / genetics
  • Sodium-Bicarbonate Symporters / chemistry*
  • Sodium-Bicarbonate Symporters / genetics

Substances

  • 3' Untranslated Regions
  • MIRN224 microRNA, human
  • MicroRNAs
  • Phosphorus Radioisotopes
  • Ribonucleotides
  • SLC4A4 protein, human
  • Sodium-Bicarbonate Symporters