Here we examined optimization of the running buffer in boronate affinity electrophoresis for improved separation of PIWI-interacting RNA (piRNA) with 2'-O-methylated ribose in 3'-terminal nucleotide. The use of Good's buffer, such as HEPES, significantly increased the separation efficiency for piRNA over normal RNA with free 3'-terminal ribose, and retained an ability to resolve the difference by at least 4-nucleotide lengths in the target piRNAs. We also demonstrated a single-step separation of piRNA from mouse testis total RNA.
- Boronate affinity electrophoresis
- Phenylboronic acid