Application of RIP-Chip for the Identification of miRNA Targets

MicroRNAs (miRNAs) are a class of noncoding small RNAs that can regulate gene expression at the posttranscriptional level. To understand how miRNAs function, it is crucial to determine the mRNA targets that are regulated by specific miRNAs. Based on known miRNA:mRNA interactions, miRNA target gene prediction programs have been developed that provide users with long lists of potential target genes. However, due to the use of different thresholds and/or criteria used, there is limited overlap between the putative miRNA targets as obtained by different miRNA target gene prediction programs. Moreover, it has been shown that there are many exceptions to the general rules of miRNA targeting, and cell-type specific miRNA and target gene expression patterns are not considered. Therefore, predicted targets need to be validated using, for instance, reporter assays that are labor intensive and may not always mimic endogenous miRNA:mRNA interactions. For these reasons, there is a clear need for a high-throughput method that allows for the unbiased detection of miRNA:mRNA interactions in the cell type of interest without the need of target gene prediction programs. Here, we provide a protocol called Ribonucleoprotein ImmunoPrecipitation – gene Chip (RIP-Chip) that results in the identification of all miRNA targets (miRNA targetome) in a given cell population. This biochemical approach is based on the immunoprecipiation of the RNA-induced silencing complex (RISC) followed by the identification of the transcripts that are enriched in the immunoprecipitated fraction by microarray analysis.