Transcriptome and targetome analysis in MIR155 expressing cells using RNA-seq

Previous studies have demonstrated the utility of microarray expression analysis to identify potential microRNA targets. Nevertheless, technical limitations intrinsic to this platform constrain its ability to fully exploit the potential of assessing transcript level changes to explore microRNA targetomes. High-throughput multiplexed Illumina-based next-generation sequencing (NGS) provides a digital readout of absolute transcript levels and imparts a higher level of accuracy and dynamic range than microarray platforms. We used Illumina NGS to analyze transcriptome changes induced by the human microRNA MIR155. This analysis resulted in a larger inferred targetome than similar studies carried out using microarray platforms. A comparison with 3′ UTR reporter data demonstrated general concordance between NGS and corresponding 3′ UTR reporter results. Nonharmonious results were investigated more deeply using transcript structure information assembled from the NGS data. This analysis revealed that transcript structure plays a substantial role in mitigated targeting and in frank targeting failures. With its high level of accuracy, its broad dynamic range, its utility in assessing transcript structure, and its capacity to accurately interrogate global direct and indirect transcriptome changes, NGS is a useful tool for investigating the biology and mechanisms of action of microRNAs.