Northern Blot Analysis of Total RNA

Total RNA was isolated using the SV Total RNA Isolation System (Promega) as described [52 (link)]. 5’ RACE was performed using the 5’/3’ RACE Kit (Roche). For Northern blotting 10–20 μg total RNA were separated using 7 M urea/10% acrylamide/0.6 x TBE gels. The RNA was transferred onto positively charged membranes (Roche) by semi dry blotting in 1 x TBE and UV crosslinked. For radioactive labeling 20 μCi γ-ATP³² (Hartmann Analytic) were mixed with 10 U T4 Polynucleotide kinase (Fermentas) and 4 pmol oligonucleotide and incubated at 37°C for 1 h. The reaction was stopped by addition of STE buffer (100 mM NaCl, 10 mM Tris, pH 8, 1 mM EDTA) and the labeled oligonucleotides were purified using MicroSpin G-25 columns (GE Healthcare). The probes were mixed with 500 μg yeast tRNA (Invitrogen) and 250 μg salmon sperm DNA (Invitrogen) to reduce unspecific probe binding. Denaturation was carried out for 10 min at 95°C. Hybridization was performed in hybridization buffer (0.5 M Na₂HPO₄, pH 7.2, 1 mM EDTA pH 7.5, 7% [w/v] SDS) over night at 68°C. The blots were washed in washing buffer (40 mM Na₂HPO₄, pH 7.2, 1 mM EDTA, pH 7.5, 1% [w/v] SDS) and exposed to a Phosphorimaging screen and analyzed with a Typhoon FLA-9000 (GE Healthcare).