title: A biochemical approach to identify direct microRNA targets.
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.
authors.affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Murugan
authors.initials: M
authors.lastname: Subramanian
authors.forename: Xiao Ling
authors.initials: XL
authors.lastname: Li
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Hara
authors.forename: Ashish
authors.initials: A
authors.lastname: Lal
mesh_terms: D016147:Genes, Tumor Suppressor; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry; D014162:Transfection / Q000379:methods*
chemical_list: C522425:MIRN34 microRNA, human; C546194:MIRN519 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D012313:RNA; D019809:Streptavidin