Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells
UMass Chan Affiliations
Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical InstituteDocument Type
Journal ArticlePublication Date
2008-09-20Keywords
AnimalsCells, Cultured
Drosophila
Gene Expression Regulation
Genetic Engineering
Humans
MicroRNAs
*Models, Genetic
Molecular Structure
Oligonucleotides, Antisense
Transfection
Life Sciences
Medicine and Health Sciences
Metadata
Show full item recordAbstract
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.Source
Nat Protoc. 2008;3(10):1537-49. Link to article on publisher's site
DOI
10.1038/nprot.2008.145Permanent Link to this Item
http://hdl.handle.net/20.500.14038/39133PubMed ID
18802435Related Resources
ae974a485f413a2113503eed53cd6c53
10.1038/nprot.2008.145