Methods for validation of miRNA sequence variants and the cleavage of their targets

MicroRNA (miRNA) variants that share the sequences with other closely related miRNAs have been identified by deep sequencing and have been implicated in the diverse regulation of their target genes. The miRNA variants that originate from the same miRNA precursor are among the most common and have been termed “isomiRs.” IsomiRs can be generated by several mechanisms such as differential processing by DICER, RNA degradation, or RNA editing. Members of the same miRNA family that have distinct sequences also contribute to the diversity of miRNA variants. Although many miRNA variants are lowly expressed and may function redundantly with their reference miRNAs, some miRNA variants are highly and/or differentially expressed. In addition, slight differences in sequence among miRNA variants can affect their specificity in target selection. Here, we describe two methods for detecting or validating miRNA variants and the target events they mediate.

► miRNA variants have been implicated in the diverse regulation of their target genes.
► Expression of variants is validated with splinted–ligation mediated miRNA detection.
► PARE is shown to be a useful tool for validating cleavage function of miRNA variants.