Short 2'-O-methyl/LNA oligomers as highly-selective inhibitors of miRNA production in vitro and in vivo.

MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors' sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We here develop a novel strategy based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Leveraging the highly conserved Xenopus miR-181a family as proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting the apical region of pre-miRNAs achieve precursor-selective inhibition of mature miRNA-5p production. Furthermore, we extend the applicability of our approach to the human miR-16 family, illustrating its universality in targeting precursors generating identical miRNAs. Overall, our strategy enables efficient manipulation of miRNA expression, offering a powerful tool to dissect the functions of identical or highly similar miRNAs derived from different precursors within miRNA families.

Droplet Digital PCR for the Detection and Quantification of Bona Fide CircRNAs.

CircRNAs are covalently closed RNA molecules gaining increasing attention over the years. Initially considered mere splicing errors, circRNAs are now recognized as a novel class of endogenous, conserved RNAs, expressed in many different species. The unique structure, the low levels of expression, and the almost complete sequence overlap with the cognate linear RNA make their detection and quantification challenging. Moreover, it has become crucial to prove the circular nature of the targeted transcript and unequivocally distinguish the circRNA from its linear counterpart. Nowadays, the most widely used technique to quantify circRNA expression is real-time quantitative PCR (qPCR). However, in the particular case of quantification of circles, it shows several technical shortcomings which affect the accuracy of the quantification. To precisely assess circRNA expression level, droplet digital PCR (ddPCR) is rapidly taking over for the more popular qPCR. In this chapter, we describe the detailed procedure based on droplets partitioning to quantify both linear and circRNA abundancy and demonstrate the circularity of the transcript under study with high precision, in a single experiment.