In vitro cultured according to published conditions
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
As per Affymetrix instructions
Hybridization protocol
Fragmentation, hybridization, washing, staining, and scanning were done according to the Affymetrix protocol. Briefly, reagent preparation included 12X MES stock (1.22 M MES, 0.89 M [Na+]) and 2X hybridization buffer (1X hybridization buffer: 100mMMES, 1M[Na+], 20mMEDTA, 0.01% Tween 20). The hybridization cocktail components and final concentrations consisted of fragmented cDNA (.05 µg/µl), control oligonucleotide B2 (50 pM), 20X eukaryotic hybridization controls bioB, bioC, bioD, and cre (1.5, 5, 25, 100 pM, respectively), herring sperm DNA(.1 mg/ml), acetylated BSA (.5 mg/ml), 1X hybridization buffer with a final volume of 300 µl. The GeneChip array was filled with 250 µl of the hybridization cocktail and hybridized for 16 hours, rotated at 60 RPM, and maintained at 45C. Reagents prepared for washing and staining included a nonstringent wash buffer (6X SSPE, .01% Tween 20), a stringent wash buffer (100mMMES, 0.1M[Na+], 0.01% Tween 20), and a 2X stain buffer (1X: 100 mM MES, 1 M [Na+], 0.05% Tween 20). The wash procedure was carried out in an Affymetrix Fluidics Station 400 controlled by Affymetrix Microarray Suite 5.0 software resident on a personal computer. The fluidics station first went through a priming step and subsequently did the washing and staining by a software protocol.
Scan protocol
The microarrays were scanned using the GeneArray scanner per manufacturer’s instructions (Affymetrix, Santa Clara, CA). The quality control algorithms for eliminating an array are based on recommendations in both the Affymetrix and dChip software packages.
Description
Gene expression data from MCF7 cells treated with 10 nM RocA for 6 Hours
Data processing
Affymetrix Microarray Suite 5.0. Microarray Suite 5.0 was used to generate a cell intensity file (*.cel). Probe intensities were normalized and summarized using GCRMA (with default settings) with R/Bioconductor.
