E-MEXP-126 - Transcription profiling of human G01518 fibroblasts starved for 16 hours in the presence of 0.1% BSA, and then stimulated for 1, 4, 10 or 24 hours with PDGF-BB (10 ng/ml in starvation medium),
Released on 30 June 2004, last updated on 2 May 2014
Subconfluent AG01518 fibroblasts were starved for 16 hours in the presence of 0.1% BSA, and then stimulated for 1, 4, 10 or 24 hours with PDGF-BB (10 ng/ml in starvation medium), or left untreated for the same periods of time. Total RNA was isolated using the RNeasy kit (Qiagen). The RNA quality was checked by formaldehyde agarose gel electrophoresis (RNeasy protocol, Qiagen). Total RNA (40 mg) from cells treated for a given period of time with PDGF or control medium were labelled in reverse transcription reactions (Superscript II kit, Invitrogen) with dCTP-Cy5 and dCTP-Cy3, respectively (Amersham). In every second replicate experiment the fluorescent deoxynucleotides were swapped. Purified cDNA probes labelled with Cy3 and Cy5 were mixed per pair, and hybridized to cDNA microarray chips (Sanger Institute H. sapiens 10K array, Hver1.2.1) from the Sanger Institute/LICR/CRUK Consortium (see http://www.sanger.ac.uk/Projects/Microarrays/ for details and hybridization protocols). For each time point, we performed at least four independent hybridizations, and used at least two different batches of RNA. Chips were scanned in a Perkin Elmer/GSI Lumonics ScanArray 4000 scanner and spot intensities were measured using the QuantArray software (histogram method with background subtraction). Normalization and statistical analysis of the quadruplicate data sets were performed using GeneSpring 5.0 analysis software (Silicon Genetics). A Lowess non-linear normalization was applied and the median of the ratio distribution for each array was set to 1. Regulated spots were selected based on the average ratio values > 1.750 for up-regulated genes and < 0.571 for down-regulated genes. In addition, we considered only genes that were significantly regulated (t-test, p<0.05) based on replicate hybridizations (global error model, GeneSpring). For all features selected using this protocol, the signal was significantly above the background, indicating that the expression of these genes was detectable. Finally, Hver1.2.1 microarrays contain replicate spots (1 to 6) corresponding to the same gene. Genes represented by spots that were not regulated in a similar manner were discarded. We show the average ratio of one representative spot for each regulated gene, with standard error calculated from multiple hybridizations and with the annotation provided by the microarray facility (Hver1.2.1_NCBI33).
transcription profiling by array, time series
Jean-Baptiste Demoulin <Jean-Baptiste.Demoulin@mexp.ucl.ac.be>, Anders Kallin, Carl-Henrik Heldin, Charlotte Rorsman, Johan Ericsson, Lars Ronnstrand
Platelet-derived growth factor stimulates membrane lipid synthesis through activation of phosphatidylinositol 3-kinase and sterol regulatory element-binding proteins. Jean-Baptiste Demoulin; Anders Kallin; Johan Ericsson; Charlotte Rorsman; Lars Ronnstrand; Carl-Henrik Heldin. J Biol Chem 279(34):35392-402 (2004)