Journal
NATURE PROTOCOLS
Volume 3, Issue 10, Pages 1537-1549Publisher
NATURE RESEARCH
DOI: 10.1038/nprot.2008.145
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Funding
- National Institutes of Health [GM62862 and GM65236]
- National Institute on Aging [F30AG030283]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R37GM062862, R01GM065236, R01GM062862] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON AGING [F30AG030283] Funding Source: NIH RePORTER
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MicroRNAs (miRNAs), B22-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.
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