Small rna marker

Pre-cast 10% polyacrylamide TBE-urea gels (Bio-Rad) were loaded with 5 µg total RNA per lane. RNA samples were mixed with TBE-urea sample buffer (Bio-Rad) and denatured at 90 °C for 5 min. On each gel, two RNA ladders were run alongside: 0.1–2 kb RNA ladder (Invitrogen) and a small RNA marker (Abnova, fragment sizes 20 to 100 bases). Gels were run for 1 to 1.5 hours at 80 V. Before blotting, lanes containing RNA ladders were cut off the gel, stained with GelRed (Biotium), and the band position documented for later RNA size estimation.
RNA was blotted onto positively charged nylon membranes (Roche) in a Mini Trans-Blot Electrophoretic Transfer cell (Bio-Rad) for 1 hour at 300 mA, then cross-linked with UV-light. 5′DIG-labelled probes were obtained from Sigma. Membranes were hybridised at 42 °C overnight at 8 rpm, with 100 ng/ml DIG-labelled probes (Table S6) in ULTRA-Hyb-Oligo hybridisation buffer (Ambion). Membrane washing and immunological detection of probes was performed using the DIG Wash and Block Buffer Set and the DIG Luminescent Detection Kit (Roche). Luminescence was recorded with BioMax Light X-ray films (Carestream). Membranes were stripped using 0.5% SDS at 60 °C for one hour and re-used for hybridisation. Northern blots were conducted in triplicate, using RNA isolated from three independent biological replicates.

Ten microliters of sample were mixed with an equal volume of gel loading buffer II (Thermo Fisher Scientific, cat# AM8546G), and the small RNA marker (Abnova, cat# R0007) was used. The mixtures were heated to 95 °C for 5 min to denature any secondary structure. Samples and markers were separated in 15% denaturing polyacrylamide gels (Invitrogen, cat# EC6885BOX) at 190 V for 50 min. Silver staining was performed using a SilverXpress Silver Staining Kit (Thermo Fisher Scientific, Cat# LC6100) according to the manufacturer’s instructions.