Hybridization solution

To validate the expression of miRNAs, northern blotting was performed according to the methods described in previous studies^{58 (link)}. In briefly, 20 μg of total RNA from each sample was separated on a 15% polyacrylamide gel and transferred to an Immobilon-Ny+ membrane (Merck Millipore, http://www.merckmillipore.com). Subsequently, the membrane was hybridized with probes labelled with c32P-ATP at 37 °C overnight in Hybridization Solution (TOYOBO, http://www.toyobo-global.com). Finally, the membrane was washed several times with low- (19 SSC, 0.5% SDS) and high-stringency (0.29 SSC, 0.2% SDS) buffer at 37 °C and exposed using a phosphorimager. The probe sequences are listed in Table S9.

RNA samples (50 μg) were denatured for 10 min at 65°C in loading buffer (TAKARA, China). The treated RNA was separated on 10% urea-polyacrylamide gels for 1.5 h at 120 V after a pre-run for 0.5 h at 200 V and transferred to Hybond-N nylon membranes (Amersham, Germany) by electroblotting for 1 h at 300 mA. Gene-specific oligonucleotides were labeled with [γ-³²P]ATP (PerkinElmer, USA) by the exchange reaction of T4 polynucleotide kinase (NEB, USA) using 10 U of enzyme, 10 pmol oligonucleotide, and 40 μCi [γ-³²P]ATP in reaction buffer for 1 h at 37°C. The membranes were UV-crosslinked at 1,200 J and the blots were prehybridized at 45°C for 1 h in Hybridization Solution (#HYB-101, TOYOBO, Japan). Hybridization with specific [γ-³²P]ATP end-labeled oligonucleotides was then performed overnight at 45°C. The membranes were washed in 0.1% SDS in 5X SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) at 45°C, followed by 0.1% SDS in 1X SSC for 30 min per wash. Signals were detected and analyzed using a Cyclone Plus Phosphor Imager (#C431200, PerkinElmer, USA). All DNA oligonucleotides used for RNA blot analysis are listed in Supplementary Table 1.