Northern Blot Analysis of milRNA

Northern blot analysis of milRNA identification was performed according to the protocol of Kim et al. with double-labeled digoxigenin (DIG) oligonucleotide probes instead of locked nucleic acids (LNA) probes [51 (link)]. Briefly, total RNA samples (5–15 ug) from the two different developmental stages were resolved on a 15% denaturing polyacrylamide gel with 8M Urea in 1X TBE. The RNA gels were then transferred to Hybond-N+ (Amersham Biosciences) at 10–15 V (30–60 min) using a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Cross-linking, hybridization and membrane detection were performed as previously described [51 (link)]. Cross-linking was performed using freshly prepared 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) reagent at 60 °C for 1 hr. Membranes were hybridized overnight in ULTRAhyb^™ hybridization buffer (Ambion) with specific double DIG-labeled oligonucleotide probes synthesized by Integrated DNA Technologies at 37 °C. Sequences of the probes used against the putative milRNAs were as follows: ccin-milR-12c, 5’-AAAGGTAGTGGTATTTCAACGGCGCC-3’; ccin-milR-13e-5p, 5’-AGTCCCTACTAGGTCCCGAG-3’. Probe detection was performed using DIG luminescent detection kit in accordance with the manufacturer’s instructions (Roche) and photoemissions were detected using the ChemiDoc-It Imaging System (Bio-rad).