ZFIN ID: ZDB-PUB-090511-13
Labeled microRNA pull-down assay system: an experimental approach for high-throughput identification of microRNA-target mRNAs
Hsu, R.J., Yang, H.J., and Tsai, H.J.
Date: 2009
Source: Nucleic acids research   37(10): e77 (Journal)
Registered Authors: Tsai, Huai-Jen
Keywords: none
MeSH Terms:
  • 3' Untranslated Regions/chemistry
  • Animals
  • Basic Helix-Loop-Helix Transcription Factors/genetics
  • Basic Helix-Loop-Helix Transcription Factors/metabolism
  • COS Cells
  • Caenorhabditis elegans/genetics
  • Caenorhabditis elegans/metabolism
  • Chlorocebus aethiops
  • Immunoprecipitation/methods*
  • MicroRNAs/isolation & purification
  • MicroRNAs/metabolism*
  • Oligonucleotide Array Sequence Analysis
  • RNA Interference*
  • RNA Precursors/isolation & purification
  • RNA Precursors/metabolism
  • RNA, Messenger/chemistry
  • RNA, Messenger/isolation & purification
  • RNA, Messenger/metabolism*
  • Zebrafish/genetics
  • Zebrafish/metabolism
  • Zebrafish Proteins/genetics
  • Zebrafish Proteins/metabolism
PubMed: 19420057 Full text @ Nucleic Acids Res.
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.