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Nucleic Acids Research Advance Access published online on May 6, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp274
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Ren-Jun Hsu, Hsin-Jung Yang and Huai-Jen Tsai*

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

*To whom correspondence should be addressed. Tel: +886 2 3366 2487; Fax: +886 2 2363 8483; Email: hjtsai{at}ntu.edu.tw

Received December 29, 2008. Revised April 10, 2009. Accepted April 10, 2009.

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.


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