Transcription profiling by array of human melanoma-derived cell lines after treatment with temozolomide

To identify patterns of gene expression that correlate with response to treatment with either melphalan or temozolomide we measured both gene expression using microarray genechips and response to drug using a standard in vitro cell proliferation assay. Senstivity to melphalan was measured 48hrs after drug treatment while sensitivity to temozolomide was measured 12 days after drug treatment. Experiment Overall Design: For each of the 50 melanoma-derived cell lines, 1 flask of cells was grown to 80% confluence and harvested for RNA isolation. Cells were lysed in buffer with 1% β-mercaptoethanol and RNA isolated using Qiagen RNeasy-plus RNA isolation kit which included a step to eliminate genomic DNA. RNA concentration was measured and quality assessed using the NanoDrop ND100 spectrophotometer. RNA was reverse transcribed and biotin-labeled cRNA synthesized. The product was hybridized to the Affymetrix human genechip HU133 Plus2 according to manufacturer's instructions. Data was initially assessed for quality and a relative value for expression was calculated as the difference between the perfect match and the mismatch signal intensities using the Affymetrix GeneChip Operating System (GCOS). A detection call (present, absent or marginal) as well as a detection p-value was also calculated. Data was normalized by multiplying each signal by a scaling factor such that the mean signal intensity for each GeneChip was equal to 500. Scaling factors were all under 10.