A biochemical approach to identify direct microRNA targets

Methods Mol Biol. 2015:1206:29-37. doi: 10.1007/978-1-4939-1369-5_3.

Abstract

We have recently developed a biochemical approach to isolate miRNA-bound mRNAs and have used this method to identify the genome-wide mRNAs regulated by the tumor suppressor miRNA miR-34a. This method involves transfection of cells with biotinylated miRNA mimics, streptavidin pulldown, RNA isolation, and qRT-PCR. The protocol in this chapter describes these steps and the issues that should be considered while designing such pulldown experiments.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Genes, Tumor Suppressor
  • HeLa Cells
  • Humans
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • RNA / isolation & purification*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • Real-Time Polymerase Chain Reaction / methods*
  • Streptavidin / chemistry
  • Transfection / methods*

Substances

  • MIRN34 microRNA, human
  • MIRN519 microRNA, human
  • MicroRNAs
  • RNA, Messenger
  • RNA
  • Streptavidin