Home  Renal Cell Carcinoma Microarray Home   Explore  Renal Cell Carcinoma Microarray Explore   Materials and Methods  Renal Cell Carcinoma Microarray Materials and Methods   Figures  Renal Cell Carcinoma Microarray Figures   Histology  H&E sections from the tumors   Supplemental data  Renal Cell Carcinoma Microarray Supplemental data   Download  Renal Cell Carcinoma Microarray downloads   Authors  Renal Cell Carcinoma Microarray Contributors		Material and Methods  cDNA clones and microarray production The 24,169 human cDNA genes/clones used in this study were obtained from Research Genetics (Huntsville AB, USA) (http://www.resgen.com/). This set of genes contained some redundancy (approximately 300 genes were printed more than once on each array) and contained approximately 4000 named genes, 2000 genes with homology to named genes in other species, and approximately 2000 ESTs of unknown function. In addition to these sequence verified clones, a small set (396) of clones whose sequence had not been verified were also included; all of these clones were excluded from all analyses presented in the figures. All of the non-sequence verified clones are identified in the primary data tables by the prefix SID (Stanford Identifier). The cDNA microarrays used in this study were made as previously described1,2. Detailed protocols are available at http://cmgm.stanford.edu/pbrown/array.html and http://cmgm.stanford.edu/pbrown/mguide/index.html. All 84 microarray experiments in this study were conducted using microarrays from a single printing. mRNA Isolations, Fluorescent cDNA Production and Hybridizations Following their excision, nephrectomy tumor samples were rapidly frozen in liquid N2 and then stored at -80 C until use. mRNA was isolated from breast tumors as described in Perou et al.3, using the Trizol Reagent (Gibco-BRL) and Invitrogen FastTrack 2.0 Kit (all Stanford samples, and see http://genome-www.stanford.edu/sbcmp/web.shtml for the detailed protocol) Common Reference Sample Each of the 44 experimental samples tested here was analyzed by a comparative hybridization, using a common "reference" mRNA pool as a standard; this reference sample was composed of equal mixtures of mRNA isolated from 11 established human cell lines (MCF7, Hs578T, OVCAR3, HepG2, NTERA2, MOLT4, RPMI-8226, NB4+ATRA, UACC-62, SW872, and Colo205: see Supplementary Information Table 1 for more details). The 11 cell lines were all grown to 70-90% confluence in RPMI medium containing 10% Fetal Calf Serum and Penicillin/Streptomycin. The cells were harvested either by scraping or centrifugation, and quickly resuspended in RNA lysis buffer and mRNA prepared as described in Perou et al.3. In each case, multiple individual mRNA preparations were collected for each cell line, which were then pooled together and analyzed via Northern analysis before final mixing to ensure the quality of the input mRNAs. The 11 mRNA samples were then mixed together in equal amounts, aliquoted in 10mM Tris (7.4), and stored at -80 C until use (2 micrograms of common reference sample was used per microarray hybridization and was always labeled using Cy3). Tumor Pathology The 44 individual kidney tumor samples used in this study were collected at either Stanford University in Stanford CA, USA, at the Palo Alto Veterans Administration Hospital in Palo Alto, CA or at the Santa Clara Valley Medical Center in Santa Clara, CA. A single pathologist (JPTH) reviewed H&E sections of each tumor, and made a histological evaluation of each. Tumors were graded using the Fuhrman grading scheme. These data are displayed in Supplementary Information Table 3 and a representative H&E section of each tumor is posted on our website at http://genome-www.stanford.edu/renal_cell_carcinoma/. Immunohistochemistry was performed as described previously3,7; the antibodies used included CD10, CK7 and vimentin. Microarray Data Analysis The cDNA microarrays were scanned with either an Axon (Foster City, CA) GenePix Scanner at 10 micron resolution. The output files, which were TIFF images, were then analyzed using GenePix software. Fluorescent ratios and quantitative data on spot quality were stored in the Stanford Microarray Database). Areas of the array with obvious blemishes were manually flagged and excluded from subsequent analyses. The primary data tables can be downloaded at http://genome-www.stanford.edu/renal_cell_carcinoma/, in text/tab delimited format after obtaining a password. Hierarchical-clustering gene selection criteria for Figure 1 (Supplementary Materials Figure 4); Data were extracted from the database in a single table, with each row representing an array element, each column a hybridization, and each cell the observed fluorescent ratio for the array element in the appropriate hybridization. This table had 1550 rows and 44 columns. Previously flagged spots were excluded. We applied average-linkage hierarchical clustering, as implemented in the program Cluster (M. Eisen; http://www.microarrays.org/software), separately to both the genes and arrays. The results were analyzed, and figures generated, using TreeView (M. Eisen; http://www.microarrays.org/software). Supplementary Information Methods References Ross, D. T. et al. Systematic variation in gene expression patterns in human cancer cell lines [see comments]. Nat Genet 24, 227-235 (2000). Alizadeh, A. A. et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling [see comments]. Nature 403, 503-511 (2000). Bindl, J. M. & Warnke, R. A. Advantages of detecting monoclonal antibody binding to tissue sections with biotin and avidin reagents in Coplin jars. Am J Clin Pathol 85, 490-493 (1986).
[ Home | Explore | Materials and Methods | Figures | Histology | Supplemental data | Download | Authors ]