For in vitro pre-miRNA processing assays, commercial recombinant Dicer enzyme (Takara) and AGO2 protein (OriGene Technologies) were used. The Dicer enzyme (2 U) or AGO2 protein (200 ng) was incubated with 20 pmol of RNA substrates in 1× reaction buffer (150 mM NaCl, 20 mM Tris-HCl (pH 8.5), 2.5 mM MgCl2) and 2 units of RNase inhibitor (Thermo Scientific). These mixtures were incubated at 37 °C for 4 h. The products were purified by phenol-chloroform extraction, followed by sodium acetate-ethanol precipitation at −20 °C.
The reaction products were separated on 7 M urea-denaturing 15% polyacrylamide gels, and then blotted onto Hybond-N + membranes (GE Healthcare) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, Berkeley, CA, USA). Hybridization was performed in Perfect Hyb solution (TOYOBO, Osaka, Japan) containing 1 ng/ml of each biotin-labelled probe (Invitrogen) for 14 h. The membranes were washed in 2× SSC with 0.1% SDS, and the signals were detected using a Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific) according to the manufacturer’s protocol. The probes labelled with biotin in this study were as follows: anti-miR-130b probe (5′-bio-TTGCCCTTTCTTCTTTGCTCTG-3′) and anti-let-7a-3 probe (5′-bio-TTCTTTTC TTCCTTCTTCCTCT-3′).
The reaction products were separated on 7 M urea-denaturing 15% polyacrylamide gels, and then blotted onto Hybond-N + membranes (GE Healthcare) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, Berkeley, CA, USA). Hybridization was performed in Perfect Hyb solution (TOYOBO, Osaka, Japan) containing 1 ng/ml of each biotin-labelled probe (Invitrogen) for 14 h. The membranes were washed in 2× SSC with 0.1% SDS, and the signals were detected using a Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific) according to the manufacturer’s protocol. The probes labelled with biotin in this study were as follows: anti-miR-130b probe (5′-bio-TTGCCCTTTCTTCTTTGCTCTG-3′) and anti-let-7a-3 probe (5′-bio-TTCTTTTC TTCCTTCTTCCTCT-3′).