RNA Isolation and Analysis of Transcripts

Total yeast RNA was purified as described [109] (link). Primer extension analysis was performed exactly as described (http://labs.fhcrc.org/hahn/Methods/mol_bio_meth/primer_ext.html) [110] (link) with the following modifications. Total RNA used was 30 µg, and in the case of more dilute RNA samples, volumes of reactions were increased by 50% to accommodate greater volume of sample. Reverse transcriptase (RT) was M-MLV RT from either Life Technologies or Fermentas. RT synthesis reactions were supplemented with 1 µl RNAse Inhibitor (Fermentas). Extension products were separated on either 7% or 10% acrylamide-bisacrylamide gels (19∶1 ratio) containing 1× TBE and 7 M Urea. Northern blotting was performed essentially as described in manual for GeneScreen hybridization membranes (Perkin-Elmer) with the following modifications. RNA samples (20 µg) were prepared in NorthernMax loading buffer (Ambion/AB). Prehybridization solution did not contain SSPE or SSC buffers, but contained 5× Denhardt's solution, 50 mM Tris-HCl pH 7.5, 1 M NaCl, 0.1% sodium pyrophosphate, 0.1% SDS instead of 1%, 10% Dextran Sulfate, 50% formamide, and 500 µg/ml sheared/denatured salmon sperm DNA. Probes for northern blots were radiolabeled using α-³²P-dATP by random priming using the Decaprime II kit (Ambion) according to manufacturer's instructions. Blots were washed at twice for 10 minutes each wash at 42°C with 2× SSC, 0.5% SDS, then twice at 67°C with 5× SSC, 0.5% SDS for 30 minutes each wash, then twice in 0.2× SSC for 30 minutes each wash at room temperature. Primer extension gels and northern blots were visualized by phosphorimaging (GE Healthcare or Bio-Rad) and quantified using ImageQuant 5.0 (GE) or Quantity One (Bio-Rad) software, with data exported to Microsoft Excel for management. Oligo sequences for site-directed mutagenesis, primer extension analysis, amplification of DNA for Northern blotting and in vitro transcription are available upon request.