Es (372); (e) other probes (843); and (f) controls. HEEBO microarrays were produced at the Stanford Functional Genomic purchase Abamectin B1a facility (Stanford, USA).Rajski et al. BMC Medicine 2010, 8:1 http://www.biomedcentral.com/1741-7015/8/Page 14 ofComplete details regarding the clones on the arrays may be found at Stanford functional genomics facility website [73]. For microarray experiments, 8 g amplified RNA (aRNA) were mixed with doping controls. Samples were vacuum dried, resolved in coupling buffer and labelled with Cy5 dye. Labelled samples were pooled with equal amounts of reverse coloured Cy3 labelled amplified reference RNA from Stratagene (Stratagene, CA, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28154141 USA). The labelled aRNA was purified with AminoAllyl MasageAmpTM II aRNA Amplification Kit (Ambion) according tothe user manual and fragmented using fragmentation reagents (Ambion). The fragmented probe was added to a hybridization buffer containing Cot/PolyA/tRNA (0.05 g/uL each), 0.3 SDS, 3.3 ?SSC and supplemented with HEPES buffer. Following a denaturing step at 100?C, the probe was placed on the microarray for competitive hybridization. After 18 h, slides with hybridized probes were sequentially washed and immediately dried in an ozone free environment and scanned using an Axon Scanner 4100A (Axon Instruments, CA, USA). The gene expression profiles of primary fibroblasts, together with accompanying clinical data are available on SMD database papers’ webpage [39]. In addition, the raw data have been deposited in NCBI’s Gene Expression Omnibus [74] and are accessible through GEO Series accession number GSE18955 http://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE18955.Data analysis and clusteringvalue of non-treated samples from each gene expression value for each cell type separately. This was performed in order to highlight those genes whose expression level changed upon treatment. Extraction of fibroblast gene signature and differentially expressed clusters was based on the correlation within the cluster nodes and, therefore, not randomly selected or based on an arbitrary cut- off. In order to compare the gene expression profile of CCL-171 and MCF-7 cells in response to IGF-I, we merged the filtered, standardized gene expression profiles of both cell lines. We then manually excluded samples with a high standard deviation between the biological replicates and those missing gene expression data. Gene expression data for different clones representing one gene were averaged. A set of 566 unique genes was hierarchically clustered in an unsupervised manner [75] and displayed using Treeview software [41].SAMFor primary fibroblasts, two-class SAM was applied [43]. One class was formed by normal and carcinoma associated fibroblasts starved in low serum medium and the other by the same cells treated with IGF-I. In order to increase the sensitivity, we paired our samples.Human cancer datasetsMicroarray fluorescent image analysis was performed using the software Genepix Pro version 5.0 3.0.6.89 (Axon Instruments). Spots with obvious array artifacts or poor technical quality were manually removed from any further analysis. Raw data files were stored in the Stanford Microarray Database [39]. The data used for the paper are available at the accompanying website at Stanford Microarray Database [39]. Data were expressed as the log2 ratio of fluorescence intensities of the sample and the reference for each element on the array. A sequential data filtering procedure was applied to include only measurem.
