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  • Total RNA was extracted from each frozen tumor specimen, and biotinylated cRNAs were generated using Trizol (Invitrogen, Carlsbad, California, United States) according to the manufacturer's instructions. Eighty-four tumor samples (including six replicates) were hybridized overnight to U133A oligonucleotide microarrays (Affymetrix, Santa Clara, California, United States), which included approximately 22,000 probe sets. In four duplicate cases two aliquots of RNA were separately used for target preparation and subsequent analysis, and in two cases, the same source cRNA was used in two independent hybridizations. Arrays were subsequently developed with phycoerythrin-conjugated streptavidin (SAPE) and biotinylated anti-streptavidin, and scanned to obtain quantitative gene expression levels. The raw gene expression values were scaled to account for differences in global chip intensity using MAS software (Affymetrix). Four scans (two duplicates and two unique tumors) were excluded because of poor quality. In total, 76 unique tumor samples were used for the analysis.

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