Yeast Total RNA Northern Blotting

Northern blotting was performed essentially as described [132 (link)]. In brief, 20 μg of yeast total RNA was prepared in Glyoxal sample load dye (Ambion) and separated by 1% agarose gel electrophoresis. RNA was transferred on to membrane by capillary blotting for pre-hybridization. Pre-hybridization solution contained 50% formamide, 10% Dextran sulfate, 5× Denhardt’s solution, 1 M NaCl, 50 mM Tris-HCl pH 7.5, 0.1% SDS, 0.1% sodium pyrophosphate, and 500 μg/ml denatured salmon sperm DNA. DNA double-stranded probes were generated by PCR and radiolabeled with ³²P-dATP using the Decaprime II kit (Ambion) according to the manufacturer’s instructions. Blots were hybridized over night at 42 °C and washed twice each in 2× SSC for 15 min at 42 °C, in 5× SSC with 0.5% SDS for 30 min at 65 °C, and in 0.2× SSC for 30 min at room temperature. Blots were visualized by phosphorimaging (Bio-Rad or GE Healthcare) and quantified using Quantity One (Bio-Rad).