Northern blotting for RNA detection

Northern blotting was performed as described in Kruszka et al. (66 (link)). Briefly: 30 µg of RNA (per sample) isolated from transfected tobacco leaves was loaded on 8 M denaturing urea polyacrylamide gel (15%) in TBE buffer (0.089 M Tris, 0.089 M boric acid, and 0.002 M EDTA, pH 8.0). RNA was then transferred onto the Amersham Hybond-NX nitrocellulose membrane (GE Healthcare) using a Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) and fixed using CL-1000 UV Crosslinker (UVP). Prehybridization and hybridization were performed in hybridization buffer (3.5% SDS, 0.375 M sodium phosphate dibasic, 0.125 M sodium phosphate monobasic) at 42 °C with DNA oligo probes (Sigma) labeled at their 5′ ends with γ³²P ATP (Hartmann Analytic). U6 was used as a loading control. After washing, the blots were exposed for up to 3 d to a phosphorimaging screen (Fujifilm) and the results were visualized with the Fujifilm FLA5100 reader (Fujifilm) and quantified using Multi Gauge V2.2 (Fujifilm).