Northern Blot Analysis of Small RNAs

10 μg RNA from total extracts was denatured at 95ºC for 10 minutes in sRNA loading buffer [20 mM HEPES pH7.8, 1 mM EDTA, 50% formamide, 3% glycerol and 0.01% bromophenol blue (BPB)], and separated in a denaturing 18% polyacrylamide gel. The RNA was blotted on an Amersham Hybond-NX nylon membrane (GE Healthcare Life Sciences) and crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 2 h at 60ºC (39 (link)). Radioactively end-labeled probes were hybridized to the membrane in PerfectHyb^TM Plus Hybridization buffer (Sigma) at 42ºC overnight. Membranes were washed 3x with 2xSSC, 2% SDS at 42ºC for 15 minutes, and developed by phosphorimaging.