Method for high confidence prediction of miRNA–mRNA targeting relationships. HITS-CLIP reads for mRNA fragments from all our time-points were pooled for the purpose of predicting miRNA/mRNA targeting relationships in the liver. Over 228 million raw sequence reads were aligned to RefSeq mRNAs to yield over 8 million fragment start positions. These mRNA fragments were coalesced into ‘footprints’ anchored by the locally strongest accumulation of reads, yielding 472,474 mRNA footprints. These footprints represent the mRNAs that are targeted to the argonaute-containing RISC at any time during liver regeneration. Next, these mRNA footprints were intersected with all possible predictions of miRNA/mRNA targeting relationships obtained from miRanda , yielding 125,949 unique and high confidence miRNA/mRNA pairs. Note that the combination of the computational approach (miRanda) with the experimental approach (HITS-CLIP) refined the computational predictions by more than 20-fold. Circles not drawn to scale.
Schug et al. BMC Genomics 2013 14:264 doi:10.1186/1471-2164-14-264