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Nucleic Acids Res. 2009 Jun;37(10):e77. doi: 10.1093/nar/gkp274. Epub 2009 May 6.

Labeled microRNA pull-down assay system: an experimental approach for high-throughput identification of microRNA-target mRNAs.

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1
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan.

Abstract

We developed a simple, direct and cost-effective approach to search for the most likely target genes of a known microRNA (miRNA) in vitro. We term this method 'labeled miRNA pull-down (LAMP)' assay system. Briefly, the pre-miRNA is labeled with digoxigenin (DIG), mixed with cell extracts and immunoprecipitated by anti-DIG antiserum. When the DIG-labeled miRNA and bound mRNA complex are obtained, the total cDNAs are then subcloned and sequenced, or RT-PCR-amplified, to search for the putative target genes of a known miRNA. After successfully identifying the known target genes of Caenorhabditis elegans miRNAs lin-4 and let-7 and zebrafish let-7, we applied LAMP to find the unknown target gene of zebrafish miR-1, which resulted in the identification of hand2. We then confirmed hand2 as a novel target gene of miR-1 by whole-mount in situ hybridization and luciferase reporter gene assay. We further validated this target gene by microarray analysis, and the results showed that hand2 is the top-scoring among 302 predicted putative target genes. We concluded that LAMP is an experimental approach for high-throughput identification of the target gene of known miRNAs from both C. elegans and zebrafish, yielding fewer false positive results than those produced by using only the bioinformatics approach.

PMID:
19420057
PMCID:
PMC2691847
DOI:
10.1093/nar/gkp274
[Indexed for MEDLINE]
Free PMC Article
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