Dr. Kapil Mehta (MD Anderson Cancer Center, Houston, TX)
Treatment protocol
MCF-7 cells were maintained in DMEM supplemented with 10 percent fetal calf serum, penicillin (100 units/mL), and streptomycin (100 ug/ml)
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from approximately 1 million MCF-7 and MCF-7/ADR cells grown in two six-well plates. The medium and any detached cells were first removed from the wells, and total RNA was isolated from the cells that remained attached to the culture dish by use of the Qiagen RNeasy kit (Valencia, CA) by the manufacturers protocol. RNA was quantitated by use of a NanoDrop micro-volume spectrophotometer (Thermo Scientific, Wilmington, DE) and then the integrity of the mRNA was verified with an Agilent BioAnalyzer (Santa Clara, CA).
Label
Biotin
Label protocol
Isolated total RNA from the MCF-7 parental cells and MCF-7/ADR cells was used with the GeneChip Sample Cleanup Module to prepare biotinylated eukaryotic complementary RNA to hybridize to the GeneChip expression probe arrays, according to the manufacturers protocol (Qiagen).
Hybridization protocol
The biotinylated complementary RNA was fragmented for 35 minutes at 94C in fragmentation buffer from the labelling kit (Enzo, Farmingdale, NY) and then hybridized to the Affymetrix GeneChip U133A 2.0 arrays (Santa Clara, CA) for 16 hours at 45C. Each probe array was then washed and stained before being scanned.
Scan protocol
Each probe array was scanned twice with the GeneArray Scanner, according to the manufacturers procedures (Affymetrix, Santa Clara, CA).
Description
MCF 7 parental cell line Control biological replicate 2
Data processing
The CEL files for hybridizations of the MCF-7/ADR cells and the MCF-7 cells were log2-transformed by use of RMA Express (Bioinformatics 2003 19(2):185-193 and Biostatistics 2003 4(2):249-264). Microarray data for the expression of 14,500 genes from MCF-7 and MCF-7/ADR were filtered with BRB-ArrayTools (version 3.8.0) (developed by Dr. Richard Simon and BRB-ArrayTools Development Team from the Biometric Research Branch at the National Cancer Institute, NIH, Bethesda, MD). The array data were filtered by use of the exclusion parameters, spot intensities that were less than 10 and genes with a P value for the log-ratio variation that was greater than .01, and a total of 4976 genes were identified for further analysis. These 4976 genes were clustered by use of the Euclidean distance and complete linkage (Supplementary Figure 1, available online). The resulting dendrogram indicated that, for all experimental conditions, the duplicate microarrays form clusters, suggesting that the results were reproducible. A two-sample t test (with random variance model) was used to perform a class comparison and determine the statistically significant genes expressed between the MCF-7 and MCF-7/ADR cells. The univariate test random variance model parameters were a equals 1.68165, b equals 14.3422, and Kolmogorov–Smirnov statistic equals 0.03998. The nominal statistical significance level of each univariate test was .001. The first 419 genes listed were statistically significant at the nominal .001 level in the univariate test with a spot intensity fold change of 10. Genes with a spot intensity fold increase of greater than 10 are shown in Supplementary Table 1 (available online), and genes with spot intensity fold decrease of greater than –10 are shown in Supplementary Table 2 (available online).
