Pri-miRNA Processing in P19 Cells

Pri-miRNA substrates were synthesized from PCR-generated DNA templates by T7 RNA polymerase in the presence of [α-³²P]UTP using a MAXIscript kit (Ambion). Nuclear extracts of P19 cells were prepared by Nuclear Extract Kit (ACTIVE MOTIF) and dialyzed in a buffer containing 20 mM HEPES (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 0.4 mM PMSF, 0.5 mM DTT and 20% (v/v) glycerol. In vitro processing assays was performed as described^{38 (link)} with some modifications. Briefly, reactions were carried out in 25 μl mixtures containing 50% (v/v) nuclear extract, 20% (v/v) reticulocyte lysate reaction mixture, 10 mM HEPES (pH 7.9), 2 mM MgCl₂, 1 mM ATP, 20 mM creatine phosphate, 50 ng yeast tRNA, 2 mM DTT, 2% polyethylene glycol (M.W. 3,550), and 100,000 c.p.m. of each pri-miRNA probe for 30 min at 30 °C. The reaction mixtures were then extracted with phenol-chloroform and the RNAs were precipitated with ethanol. The RNA samples were resolved on a urea-6% polyacrylamide TBE gel (Invitrogen) with Dynamarker Small RNA plus as a size marker and exposed to X-ray film (Kodak) at −80 °C.