NCI-60 Cancer Cell Line transcript expression profiling on HG-U133A GeneChip® arrays (Affymetrix, Santa Clara, CA, USA) were performed.
Growth protocol
The protocols for cell culture, cell harvests, and RNA purification, and microarray studies have been described in detail elsewhere (Shankavaram, et al., manuscript in preparation). Briefly, seed cultures of the 60 cell lines were drawn from aliquoted stocks, passaged once in T-162 flasks, and monitored frequently for degree of confluence. The medium was RPMI-1640 with phenol red, 2 mM glutamine, and 5% fetal bovine serum. For compatibility with our other profiling studies, all fetal bovine serum was obtained from the same large batches as were used by DTP for the drug screen. One day before harvest, the cells were re-fed. Attached cells were harvested at ~80% confluence, as assessed for each flask by phase microscopy. Suspended cells were harvested at ~0.5x106 cells/mL. In pilot studies, samples of medium showed no appreciable change in pH between re-feeding and harvest, and no color change in the medium was seen in any of the flasks harvested. The time from incubator to stabilization of the preparation was kept to <1 min. Total RNA was purified using the Qiagen (Valencia, CA) RNeasy Midi Kit according to manufacturer's instructions. The RNA was then quantitated spectrophotometrically and aliquoted for storage at –80oC.
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 microg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following fragmentation, 10 microg of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome Array HG-U133A. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
Description
Gene expression data from NCI-60 cancer cell lines before drug treatment.
Data processing
The data were analyzed with RMAexpress (Robust Microarrya Analysis) using its default analysis settings and quantile normalization method.
