Perfecthyb plus hybridization buffer liquid

A total of 60 µg total RNA was loaded onto a 14% denaturing urea-polyacrylamide gel and run with 0.5x TBE at 150V. The gel was transferred to Amersham HybondTM-NX with 14A overnight. Chemical cross-linking buffer was prepared as follows: 0.373 g N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (Sigma-Aldrich, E7550, St. Louis, Missouri, MI, USA), 3 drops of 1 M HCl, 121 µl 1-Methylimidazole (Sigma, M50834), and 12 mL DEPC-treated ddH2O. Membrane was putted on a plate which has two layers of filter paper that was soaked in cross-linking buffer, then the plate was covered by plastic film andincubatedat60 °C for 2 h and at 85 °C for another2 h. The probes were labeled withγ-P32 ATP by T4-polynucleotide kinase (NEB). Pre-miRNA Northern blot probes are the same as miRNA Northern blot probes. The membrane was pre-incubated with PerfectHyb™ Plus Hybridization Buffer liquid (Sigma) for 30min, then the labeled probe was added and incubated for 12h. After that, the membrane was wash with buffer contains 2x SSC and 0.025% SDS for four times. Signal was collected with phosphor screen and scanned with typhoon. Similar results were obtained from three biological repeats.

Northern blot was performed as described [35 , 84 ]. Total RNA was separated on 14% denaturing urea-polyacrylamine gels and run with 0.5 × TBE at 150 V. The gel was transferred to Hybond membrane NX (GE healthcare, RPN303T) at 14 V overnight. Chemical crosslink buffer was prepared as follows: 0.373 g N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (Sigma-Aldrich, E7550), 3 drops of 1 M HCl, 121 µl Methylimidazole (Sigma-Aldrich, M50834), and 12 mL RNase-free H₂O. After chemical crosslink at 60 °C for 2 h and UV crosslink at 85 °C for 2 h, the membrane was pre-incubated with PerfectHyb™ Plus Hybridization Buffer liquid (Sigma-Aldrich, H7033) for 30 min, then hybridized overnight at 37 °C with γ- ³²P ATP (China isotope & radiation corporation) labelled DNA probes by T4-polynucleotide kinase (NEB, M0201S) for 4 h. After that, the membrane was washed with buffer contains 2 × SSC and 0.025% SDS. Auto-radiography of the membrane was performed using a Typhoon Scanner. Sequences of probes are listed in Supplementary Table S4.