title: Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells.
abstract: MicroRNAs (miRNAs), approximately 22-nt RNAs that mediate post-transcriptional regulation of mRNAs in animals and plants, are a diverse class of regulatory genes whose specific biological functions are largely unknown. Here we detail a protocol to design and introduce into cultured Drosophila and human cells sequence-specific antisense oligonucleotides (ASOs) that block the function of individual miRNAs. Coupled with recent studies that catalog the miRNAs expressed in diverse cultured cells, our method offers a rapid (<1 week) approach to validate miRNA targets and to study the cellular functions of individual human and Drosophila miRNAs. ASO-based inactivation of miRNAs is faster and simpler than comparable genetic or 'sponge'-based approaches, for which extensive recombinant DNA manipulation is required. We present our ASO design principles and an optimized transfection protocol in which transfection efficiency of Drosophila Schneider 2 cells can approach 100%. Our 3'-cholesterol-modified ASOs have enhanced potency, allowing miRNA inhibition for at least 7 d from a single transfection.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Horwich
authors.forename: Phillip D
authors.initials: PD
authors.lastname: Zamore
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004330:Drosophila; D005786:Gene Expression Regulation / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D008957:Models, Genetic; D015394:Molecular Structure; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D035683:MicroRNAs; D016376:Oligonucleotides, Antisense