A rapid and simple method for preparation of RNA fromSaccharomyces cerevisiae

Abstract

Most methods for isolation of RNA from yeast require tedious vortexing with glass beads, and give low yields when scaled down to 10 ml cultures (1). In addition, it is frequently desirable to prepare RNA from several different yeast strains grown under a variety of growth conditions, and preparations using glass beads are impractical when dealing with multiple samples.Heating and freezing of yeast cells in the presence of phenol and SDS has been used for large scale isolation of RNA from yeast (2). We report here a mini-prep version of yeast RNA isolation using phenol and SDS that allows for the processing of a dozen samples in about 60 minutes, and provides enough RNA to run several northern blots. In our hands methods using glass beads are slower and give lower yields of RNA.RNA was isolated as follows. Ten ml cultures were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD6W of 2.5-5.0. We have also used YP 2% galactose, YP 4% ethanol/3 % glycerol, YP 2% raffinose and a variety of minimal media. The cells were harvested by centrifugation and resuspended in 400 td of 50 mM Na acetate pH 5.3, 10 mM EDTA ('AE buffer').The resuspended cells were transferred to a 1.5 ml microcentrifuge tube and 40 IAI of 10% SDS was added. The suspension was vortexed and an equal volume of fresh phenol, previously equilibrated with AE buffer, was added. The mixture was again vortexed and incubated at 65°C for 4 min. The mixture was then rapidly chilled in a dry ice/ethanol bath until phenol crystals appeared, and then centrifuged for 2 min at maximum speed in a microcentrifuge to separate the aqueous and phenol phases. The upper, aqueous phase was transferred to a fresh microcentrifuge tube and extracted with phenol/chloroform at room temperature for 5 min. The extracted aqueous phase was then brought to 0.3 M Na acetate, pH 5.3, by adding 40 tl of 3 M Na acetate pH 5.3, after which 2.5 volumes of ethanol were added to precipitate the RNA. After washing with 80% ethanol, the pellet was dried and resuspended in 20 i1 of sterile water and stored at -70°C until used. Throughout the preparation, normal precautions to avoid ribonuclease contamination were taken (3).The isolation procedure was performed using seven different wild type yeast strains and over two dozen derivatives of these strains. Yields varied between 60 ,ag and 300 Mg of RNA per 10 ml culture, with an average yield of 135 ,ug as quantitated by absorbance at 260 nm. Fig. la shows the quality of the RNA that is isolated using this method. No degradation of ribisomal RNA bands is seen, and recoveries of both high and low molecular weight RNA appear excellent.

Remodeling of Yeast Genome Expression in Response to Environmental Changes

“…Briefly, total RNA was first extracted from frozen cell pellets by a hot phenol method (Schmitt et al, 1990). Cells were thawed on ice and then mixed with 3 ml of acid phenol/chloroform (5:1; prewarmed for 10 min at 65°C) and 3 ml of TES (10 mM Tris, pH 7.5/10 mM EDTA/0.5% SDS) per 200 OD 600 U.…”

The YeastSaccharomyces cerevisiaeContains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

“…For Northern analysis, cell cultures were grown to an A 600 of 1.0, and RNA was extracted by the method of Schmitt et al (1990). Total RNA (10 g) was separated by electrophoresis in a 1% formaldehyde gel.…”

Artificially Recruited TATA-Binding Protein Fails To Remodel Chromatin and Does Not Activate Three Promoters That Require Chromatin Remodeling

“…RNA was extracted from yeast (66), and 10 g was run on agarose-formaldehyde gels (71). Gels were blotted onto nylon membranes in 10ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate), UV cross-linked, and hybridized (11) with probes labeled by random priming.…”