Tandem affinity purification of miRNA target mRNAs (TAP-Tar)

Nucleic Acids Res. 2010 Mar;38(4):e20. doi: 10.1093/nar/gkp1100. Epub 2009 Dec 2.

Abstract

MicroRNAs (miRNAs) bind to Argonaute proteins, and together they form the RISC complex and regulate target mRNA translation and/or stability. Identification of mRNA targets is key to deciphering the physiological functions and mode of action of miRNAs. In mammals, miRNAs are generally poorly homologous to their target sequence, and target identification cannot be based solely on bioinformatics. Here, we describe a biochemical approach, based on tandem affinity purification, in which mRNA/miRNA complexes are sequentially pulled down, first via the Argonaute moiety and then via the miRNA. Our 'TAP-Tar' procedure allows the specific pull down of mRNA targets of miRNA. It is useful for validation of targets predicted in silico, and, potentially, for discovery of previously uncharacterized targets.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotinylation
  • E2F1 Transcription Factor / genetics
  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • MicroRNAs / isolation & purification*
  • MicroRNAs / metabolism
  • RNA, Messenger / isolation & purification*
  • RNA, Messenger / metabolism

Substances

  • E2F1 Transcription Factor
  • MIRN20a microRNA, human
  • MicroRNAs
  • RNA, Messenger