May 22, 2014

Profiling direct mRNA-microRNA interactions using synthetic biotinylated microRNA-duplexes

BioRxiv : the Preprint Server for Biology
Shivangi Wani, Nicole Cloonan


MicroRNAs (miRNAs) are predominantly negative regulators of gene expression that act through the RNA-induced Silencing Complex (RISC) to suppress the translation of protein coding mRNAs. Despite intense study of these regulatory molecules, the specific molecular functions of most miRNAs remain unknown, largely due to the challenge of accurately identifying miRNA targets. Reporter gene assays can determine direct interactions, but are laborious and do not scale to genome-wide screens. Genomic scale methods such as HITS-CLIP do not preserve the direct interactions, and rely on computationally derived predictions of interactions that are plagued by high false positive rates. Here we describe a protocol for the isolation of direct targets of a mature miRNA, using synthetic biotinylated miRNA duplexes. This approach allows sensitive and specific detection of miRNA-mRNA interactions, isolating high quality mRNA suitable for analysis by microarray or RNAseq.

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Mentioned in this Paper

Genome-Wide Association Study
Microarray Analysis
SCPEP1 gene
Sequence Determinations, RNA
Profile (Lab Procedure)
Gene Expression
RNA-Induced Silencing Complex
RNA-induced Silencing Complex Location

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