tissue: non-small cell lung cancer cell line drug response: gefitinib-resistant cell line cell line: PC9GRM2 time: 0.5hr treatment: EGF
Treatment protocol
After 48-hr starvation, each cell line were treated with 100 ng/ml EGF and/or 0.5 uM gefitinib. Cells were harvested at 26 time points during 24 hours.
Growth protocol
Parental PC9 cells were grown in RPMI1640 media supplemented with 10% fetal bovine serum. For the stablishment of gefitinib-resistance cell line (PC9GR), PC9 cells were treated with growth medium containing gefitinib at the concentrations 0.02-0.1 uM for 14-28 days, 0.005-0.02 uM for 14-28 days, and 0.1 uM for a range of exposure durations, yielding approximately 500 survived clones. Following further treatments of gefitinib and repeated clone refining with the indicated concentrations (0 uM, 0.1 uM and 1 uM), a total of 179 clones were screened out based on MTT assays that verified significant suppression of growth in the cancer populations after 7 days. Among the clones further refined that lacked the MET amplification and the EGFR T790M mutation, PC9GR was isolated as a line demonstrating one of the lowest dose responses and almost the same doubling time to PC9.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from each sample by TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s directions. The quality of the total RNA was evaluated on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
RNA samples were labeled using the Agilent Low RNA Input Linear Amplification Kit (Agilent Technologies) according to the manufacturer’s directions. Briefly, amplification and labeling of total RNA (500 ng) was performed using Cyanine 3-CTP.
Hybridization protocol
Hybridization was carried out using the Gene Expression Hybridization Kit (Agilent Technologies), according to the manufacturer’s directions. Briefly, a cRNA sample (1.65 g) was subjected to fragmentation (30 min at 60 °C) and then hybridization on a 44K Agilent Whole Human Genome Oligo Microarray (G4112F) (Agilent Technologies). . RNA samples with an RNA integrity number (RIN) greater than 8 were used for further analysis. Hybridization was carried out on a 44K Agilent Whole Human Genome Oligo Microarray (G41112F) (Agilent Technologies).
Scan protocol
Microarrays were scanned by a dynamic autofocus microarray scanner (Agilent DNA Microarray Scanner, Agilent Technologies) under the default parameters (Green PMT was set to 100%, and the scan resolution was set to 5 um. Feature Extraction Software v9.1 (Agilent Technologies) was used to obtain a raw signal value and a quality flag (Present [P], Marginal [M], or Absent [A]).
Description
Gene expression after 0.5hr in PC9GRM2 treated with EGF
Data processing
After aggregating all the scanned signals for the different time points, cell lines, and treatments into a single data matrix, quantile normalization (Bolstad et al., Bioinformatics, 2003) was applied to remove the between-experiment distributional biases due to assay artifacts by forcing the same percentile values on all of the processed data.
Time-course gene expression profiles of gefitinib-sensitive or -resistant lung adenocarcinoma cells stimulated with EGF, in the presence or absence of gefitinib, EGF receptor tyrosine kinase-specific inhibitor
