Yeast cells were grown at 30 degrees Celsius in a shaking incubator overnight to saturation in 10mL of YPD complete media, yeast extract peptone, supplemented with 2% glucose.
Extracted molecule
total RNA
Extraction protocol
Hot phenol purification method was used to prepare the RNA. Specifically, 10mL of saturated overnight YPD cultures were resuspended in 400uL of acetate-EDTA (AE) buffer (50mM Na-acetate, pH 5.3, 10mM EDTA). 40uL of 10% sodium dodecyl sulfate (SDS) was added. The cells were vortexed before and after adding equal volumes of pre-equilibrated RT phenol. Samples were incubated at 65C for four minutes then chilled rapidly in a dry ice/EtOH bath until phenol crystals appeared. The aqueous layer was extracted with RT phenol/chloroform after centrifugation. 3M Na-acetate, pH5.3, was added to the extracted phase to a final concentration of 0.3M and 2.5 volumes of 100% EtOH was added to precipitate the RNA. After a 20 minute centrifugation, the pellet was washed with 80% EtOH then dried in a speedvac (ThermoSavant). The RNA was resuspended in 40uL of sterile water. After diluting 10-fold, the RNA was quantified by spectrophotometer (Nanodrop, ND-1000). Yields ranged from 40 to 160ug. RNA was checked by gel electrophoresis prior to microarray analysis. Samples were stored at -20C or -80C for short-term or long-term storage, respectively.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following fragmentation, 5 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Yeast 2.0 Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix GS 3000 Scanner
Description
esa1 sup2-
Data processing
The data were analyzed in GCOS using the MAS 5.0 algorithm. The data was analyized using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.
