Separation of miRNA and its methylation products by capillary electrophoresis

Methylation is a crucial step in plant microRNA biogenesis. To improve our understanding of the methylation process and its regulation, a rapid and convenient high-throughput method should be sought to help with the study of reaction kinetics and assist the search for chemical inhibitors of the methyltransferase, HEN1. In this short communication, we report a pioneering work that used capillary electrophoresis (CE) to separate the miRNA and its methylation product. Capillary zone electrophoresis (CZE) with UV-absorption detection and a reduced running temperature achieved good separation of miR173/miR173* and miR173-m/miR173*-m with a detection limit of around 1 μM. To enhance detection sensitivity, capillary gel electrophoresis (CGE) coupled with laser-induced fluorescence (LIF) detection was also tested, and base-line separation of nanomolar duplex RNA samples was achieved using 4% polyvinylpyrrolidone (PVP) as the sieving matrix and SYBR Green II RNA gel stain for on-column labeling. Although further study is needed to investigate if the separation is sequence dependent, our study demonstrated, for the first time, that CE could be an effective and rapid method in monitoring the miRNA methylation process.