A sensitive and reproducible method to validate several hundred microRNA target genes in one experiment is described online this week in Nature Methods. These results give a better understanding of how these target genes could be regulated.
MicroRNAs are short RNA molecules that do not encode a protein but are crucial in regulating gene expression by blocking translation or marking the RNA for degradation. The targets of specific microRNAs can be predicted by computational algorithms, but experimental approaches to validate the predicted targets at a large scale are needed.
Michael Hengartner and colleagues report a proteomics approach to follow up on computationally predicted microRNA target genes in the roundworm Caenorhabditis elegans. To determine which genes are regulated by a specific microRNA known as let-7, the researchers compared protein levels in normal worms to protein levels in mutant worms in which let-7 levels were reduced. They used a highly sensitive detection technique called selected reaction monitoring (SRM) mass spectrometry in combination with a quantitative method, isotope-coded affinity tagging (ICAT). When the protein levels changed in the mutant worms compared to the normal worms, this indicated that the gene was indeed regulated by let-7.
The general approach should be readily adaptable to validate the predicted target genes of any microRNA in any organism.