A single colony of strain S288C was inoculated into 10 ml of YPD (1%(w/v) yeast extract/2%(w/v) Bacto peptone/2%(w/v) glucose) and grown with shaking at 30°C for overnight. To prepare cells under a rich condition, they were resuspended in 100 ml of YPD at an OD600 of 0.1 and grown at 30°C for 6 hrs. To prepare cells under a poor condition, the cells grown overnight in YPD media were washed with ddH2O, resuspended in 100 ml of SD (0.67%(w/v) yeast nitrogen base without amino acids/2%(w/v) glucose) at an OD600 of 0.5, and grown at 30°C for 6 hrs. The cells were collected by centrifugation, resuspended in ddH2O, aliquoted in microtubes (400 μl), frozen in liquid nitrogen, and stored at -80°C until use. The number of cells was directly counted using a hematocytometer.
Total RNA was extracted using a hot-phenol method [12 (link)] with some modifications. To the 400-μl cell suspension described above, 100 μl of 5× lysis buffer (50 mM Tris-HCl, pH 7.5/50 mM EDTA/2.5%(w/v) SDS) and 500 μl of water-saturated phenol were added and mixed well on a shaker at 65°C for 1 hr. The tubes were chilled on ice for 5 min and centrifuged for phase separation. While the aqueous phase was saved in another tube, the phenol phase was mixed with 500 μl of 1× lysis buffer and shaked at 65°C for 1 hr. The second aqueous phase was combined with the first one and extracted once with water-saturated phenol and once with chloroform. The RNAs was precipitated by adding isopropanol to the aqueous phase, rinsed with 75%(v/v) ethanol, and dissolved in ddH2O. To remove contaminating genomic DNA, the RNA was treated with RNase-free DNase I (Promega) and purified with TRIzol reagent (Invitrogen) according to the manufacturer's instruction. The concentration of RNA was determined by measuring OD260 on spectrophotometer based on an assumption that one OD260 unit corresponds to 40 ng/μl of RNA.
Total amount of cellular RNAs was also determined using selective extraction of ribonucleotides by NaOH [13 (link)] with some modifications. To the 400-μl cell suspension, 100 μl of 1.2 N perchloric acid (PCA) was added and the obtained mixture was placed in ice-cold water for 1 hr. Following centrifugation, the supernatant was removed and the cell pellet was washed again with 500 μl of 0.25 N PCA. Following careful removal of residual PCA solution, the cell pellet was resuspended in 300 μl of 0.3 N NaOH and incubated at 37°C for 1 hr. After neutralization with adding 150 μl of 1.2 N PCA, the concentration of RNA was determined from OD260 and a standard curve obtained from the measurement of various known amounts of purified yeast RNA subjected to the same NaOH/PCA treatment.
Total RNA was extracted using a hot-phenol method [12 (link)] with some modifications. To the 400-μl cell suspension described above, 100 μl of 5× lysis buffer (50 mM Tris-HCl, pH 7.5/50 mM EDTA/2.5%(w/v) SDS) and 500 μl of water-saturated phenol were added and mixed well on a shaker at 65°C for 1 hr. The tubes were chilled on ice for 5 min and centrifuged for phase separation. While the aqueous phase was saved in another tube, the phenol phase was mixed with 500 μl of 1× lysis buffer and shaked at 65°C for 1 hr. The second aqueous phase was combined with the first one and extracted once with water-saturated phenol and once with chloroform. The RNAs was precipitated by adding isopropanol to the aqueous phase, rinsed with 75%(v/v) ethanol, and dissolved in ddH2O. To remove contaminating genomic DNA, the RNA was treated with RNase-free DNase I (Promega) and purified with TRIzol reagent (Invitrogen) according to the manufacturer's instruction. The concentration of RNA was determined by measuring OD260 on spectrophotometer based on an assumption that one OD260 unit corresponds to 40 ng/μl of RNA.
Total amount of cellular RNAs was also determined using selective extraction of ribonucleotides by NaOH [13 (link)] with some modifications. To the 400-μl cell suspension, 100 μl of 1.2 N perchloric acid (PCA) was added and the obtained mixture was placed in ice-cold water for 1 hr. Following centrifugation, the supernatant was removed and the cell pellet was washed again with 500 μl of 0.25 N PCA. Following careful removal of residual PCA solution, the cell pellet was resuspended in 300 μl of 0.3 N NaOH and incubated at 37°C for 1 hr. After neutralization with adding 150 μl of 1.2 N PCA, the concentration of RNA was determined from OD260 and a standard curve obtained from the measurement of various known amounts of purified yeast RNA subjected to the same NaOH/PCA treatment.
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