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PMID: 18802435 Published · ppublish English Comparative Study Journal Article Research Support, N.I.H., Extramural

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

Nature protocols ·Vol. 3 ·No. 10 ·2008-00-00 ·Pages 1537-49

Horwich MD, Zamore PD

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.

MeSH Terms
Animals Cells, Cultured Drosophila Gene Expression Regulation/genetics Genetic Engineering/methods Humans MicroRNAs/metabolism Models, Genetic Molecular Structure Oligonucleotides, Antisense/chemistry,metabolism Transfection/methods
Chemicals
MicroRNAs Oligonucleotides, Antisense
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Horwich Michael D
Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Zamore Phillip D
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Article Info
Journal
Nature protocols
Abbr.
Nat Protoc
ISSN
1750-2799
DOI
10.1038/nprot.2008.145
Published
2008-00-00
Pages
1537-49
Language
English
Region
England
NLM ID
101284307
PMCID
PMC2559958
Subset
IM
Grants
NIGMS NIH HHS · GM65236 · United States
NIGMS NIH HHS · R01 GM062862 · United States
NIGMS NIH HHS · R01 GM065236-06 · United States
NIA NIH HHS · F30 AG030283 · United States
NIGMS NIH HHS · R01 GM065236 · United States
NIA NIH HHS · F30AG030283 · United States
NIGMS NIH HHS · GM62862 · United States
NIGMS NIH HHS · R01 GM062862-07 · United States
NIGMS NIH HHS · R37 GM062862 · United States
NIA NIH HHS · F30 AG030283-02 · United States
External Links
PubMed source DOI: 10.1038/nprot.2008.145 PMC Full Text View journal details
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