miRNA Isolation from Embryonic Tissues

Total RNA was extracted from 0.5 mg of embryonic midbrain tissues and from H19-7 cells (10² to 10⁷ cells) and using the mirVana®miRNA Isolation kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions and as previously described by Kerek et al. [14 (link)]. miRNAs were isolated using a two-step procedure. In the first step, samples were disrupted in a denaturing lysis buffer and then subjected to acid-phenol/chloroform extraction. The second step consisted on purification over glass-fiber filter that immobilizes the RNA which was later eluted using RNase-free water. According to the manufacturer’s instructions, no enrichment procedure is needed while isolating miRNA for expression profiling using miRNA arrays. The concentration and purity of RNA were determined at 260/280 nm by using a nanodrop spectrophotometer (Multiskan GO).