High-throughput isolation of Saccharomyces cerevisiae RNA.

The yeast Saccharomyces cerevisiae has long been an important model organism for biological investigation (1). More recently, high-throughput approaches using deletion libraries and fusion protein libraries have provided powerful techniques with which to study the entire yeast genome and proteome (2–6). However, the methods typically used for preparing high-quality RNA samples from yeast are impractical to perform in a high-throughput format. These techniques involve either hot phenol extraction, mechanical disruption with glass beads, or lyticase treatments to break the cell wall and/or require additional organic extractions and alcohol precipitations to isolate the RNA (7–9). Handling large amounts of phenol poses safety risks, the mechanical lysis of yeast cells becomes tedious when working with large numbers of samples, and the multiwell plates typically used in high-throughput screens cannot be centrifuged at the high speeds required for RNA precipitation. We have developed a method for isolating S. cerevisiae RNA in a 96-well format conducive to high-throughput studies. In this method, RNA is released from the cell upon digestion with proteinase K, and proteins and cell debris are precipitated with potassium acetate under low-speed centrifu-gation. RNA is then purified from the cleared extract using a commercially available nucleic acid binding plate in conjunction with a vacuum manifold. To perform this method, S. cerevisiae cultures were grown 1 to 2 days in 0.5 mL selective medium in a 2-mL deep-well 96-well plate sealed with air-permeable tape. Cells were collected by centrifugation at 1200× g for 5 min (approximately 2500 rpm in a Beckman GS-6R centrifuge equipped the supernatants were discarded by aspiration, leaving approximately 25 µL liquid/well. The cell pellets were resus-pended in the residual liquid by vortex mixing, and 300 µL proteinase K buffer [10 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, 150 mM NaCl, 1% sodium dodecyl sulfate (SDS), 0.4 mg/mL proteinase K (Sigma, St. Louis, MO, USA)] prewarmed to 65°C were added to each well and mixed thoroughly by gentle vortex mixing. Vigorous vortex mixing was avoided, as this can cause splashing, particularly with larger volumes. Protease digests were incubated for 15 min in a 65°C water bath with periodic mixing and then placed on ice for 20 min. Next, 175 µL ice-cold KOAc precipitation solution [3 M potassium acetate, 11.5% (v/v) glacial acetic acid] were added to each well, and the plate was vortex mixed for 10 s. Following centrifugation at 1650× g for 20 min, the supernatants …