Dynamic response of EGF stimulation in lung cancer cells [EGF]

The analysis of microarray time series promises a deeper insight into the dynamics of the cellular response following stimulation. A common observation in this type of data is that some genes respond with quick, transient dynamics, while other genes change their expression slowly over time. The existing methods for the detection of significant expression dynamics often fail when the expression dynamics show a large heterogeneity, and often cannot cope with irregular and sparse measurements. H1975 NSCLC cells were seeded in 6-well-plates with 1,33x10(-5) cells per well. After incubation for 3 days, cells were washed 3 times and supplemented with medium without FCS for overnight starvation. The following day, cells were stimulated with 50 ng/ml of EGF diluted in starvation medium. Samples were harvested after 0, 0.5, 1, 2, 4, 6, 8, 12, 24 and 48 hours. Total cellular RNA was isolated with the NucleoSpin RNA II kit according to the manufacturers' instructions. After amplification, labeling with biotin and fragmentation of the RNA, hybridization with GeneChip Human Gene 2.0 ST Array was performed for 16 h at 45°C. Subsequently, washing and staining was performed using an Affymetrix Fluidics Station 450 and the microarray was scanned using an Affymetrix GeneArray Scanner 3000.