Control cells received no gentamicin, Gent treated cells were acclimated to growth in gentamicin, 500ug/ml and grown with gentamicin throughout the experiment
Growth protocol
Cell were grown overnight in YPD +/- gentamicin, diluted to 5x106 cells/ml and grown to mid-log with and without gentamicin 500ug/ml
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the Epicentre Masterpure Yeast RNA Purification kit (Madison,WI)
Label
biotin
Label protocol
Protocols recommended by Affymetrix were used, cDNA was synthesized using a T7 promoter-dT24 oligonucleotide as primer with the Invitrogen Life technologies SuperScript Choice system. Following second strand cDNA synthesis and incubation with T4 DNA polymerase, the products are purfied using an Affymetrix Cleanup Module. Biotinylated cRNA is made using the Affymetrix IVT kit. The cRNA is purified using Qiagen RNeasy columns, quantitated and then fragmented by incubation at high temperature with magnesium.
Hybridization protocol
Fifteen ug of biotinylated cRNA is added to a total hybridization cocktail of 300ul, and 200ul is hybridized to a GeneChip after adding control oligonucleotides. Hybridization is at 45C for 17 h with constant rotation. The hybridization mixture is then removed and the GeneChips are washed, stained with phycoerythrin-labeled Streptavidin, washed, incubated with biotinylated anti-strepavidin, and then restained with phycoerythrin-labeled Strepavidin to amplify the signals; these steps are carried out using the Affymetrix Fluidics Station. To reduce non-random error, balanced groups of samples are handled in parallel.
Scan protocol
Arrays are then scanned using the dedicated scanner, controlled by Affymetrix GCOS software. Images are examined for defects.
Description
control
Data processing
The Affymetrix Microarray Suite version 5 (MAS5) algorithm analyzes the hybridization intensity data from GeneChip expression probe arrays and calculates a set of metrics that describe probe set performance. The average intensity on each array is normalized by global scaling to a target intensity of 1000.
