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Nat Protoc. 2008;3(10):1537-49. doi: 10.1038/nprot.2008.145.

Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells.

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1
Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

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.

PMID:
18802435
PMCID:
PMC2559958
DOI:
10.1038/nprot.2008.145
[Indexed for MEDLINE]
Free PMC Article
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