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

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.

miR-1 and miR-206 target different genes to have opposing roles during angiogenesis in zebrafish embryos

“…Using the LAMP assay system and microarray analysis, we have previously searched all the putative target genes of mature miR-1 (ref. 18). Here, we went further to focus on the candidate genes which are expressed in the muscle during zebrafish embryogenesis.…”

“…As no consensus has thus far been reached with respect to the regulatory function(s) of miR-1/ 206, we conducted a more intensive examination of the molecular mechanism underlying the activity of these muscle-specific miRNAs in modulating angiogenesis during zebrafish embryonic development. To accomplish this, we employed the labelled miRNA pull-down (LAMP) assay system 18 to obtain the target genes specific for miR-1 and miR-206.…”

MicroRNAs in Vascular and Metabolic Disease

“…The LAMP assay is an in vitro approach that utilizes labeling of the pre-miRNA with digoxigenin (DIG). 98 The DIG labeled precursor is mixed with cell extracts and immunoprecipitated by anti-DIG antibody. The obtained DIG-labeled miRNA and bound mRNA complexes are isolated and the complementary DNAs are subcloned and sequenced or amplified by reverse transcription polymerase chain reaction to search for putative target genes of the miRNA.…”

MicroRNA Screening and the Quest for Biologically Relevant Targets

“…114 A similar approach has been applied for miRNA target discovery using digoxigenin-labeled precursor miRNAs. 117 An advantage of this approach over other techniques based on miRISC immunoprecipitation is the enrichment for targets of the biotin-modified transfected miRNA. However, as for other protocols described previously, caveats associated with miRNA overexpression should be taken in consideration, as well as concerns associated with the introduction of a tag in the miRNA, which may affect the profile and specificity of miRNA-mRNA binding.…”