miRNA Trapping by RNA in Vitro Affinity Purification
miTRAP identifies the target RNA sequences of miRNA species in vitro utilizing MS2 stem loops attached to the target RNA sequence as bait for the miRNA (Braun et al., 2014). MS2 is a 19 nt bacteriophage RNA sequence present at the ribosomal binding site of the MS2 replicase mRNA that folds into a hairpin loop structure.
First, MS2 bait transcripts are transcribed in vitro. The miRNA-protein-RNA complexes are captured by immobilization on amylose resin. The RNA-protein complexes are incubated with cellular extracts, and the extracted RNA-protein complexes are eluted using maltose solution. miRNAs are purified from the maltose solution by a phenol-chloroform extraction, while protein analysis is carried out after incubating the resin with SDS-sample buffer containing 10% _-mercaptoethanol. miRNA eluates are prepared into cDNA libraries, using standard small-RNA Library Prep Kit, and sequenced.
Similar methods: CLASH
- Enables in vitro identification of miRNA targets
- Also identifies novel and noncanonical miRNA target sequences involved in gene regulation
- Not yet validated for analyzing trans-acting RBPs or lncRNAs that affect miRNA targeting
- Unable to incorporate UV or chemical-based protein-RNA crosslinking prior to cell lysis
Illumina Library prep and Array Kit Selector
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