The sample preparation methods include isolation of total RNA, then linear amplification and preparation of biotinylated cRNA, following the recommendations of the GeneChip Expression Analysis Technical Manual. Gene Logic's molecular biologists optimize the methods when necessary to ensure that high quality data is generated from each specimen. Biotin-labeled cRNA is fragmented prior to hybridization on the Affymetrix Human Genome U133 GeneChip array, the Murine Genome U74v2 Set, or the Rat Genome U34 Set. The arrays are hybridized for 24 hours at 45oC, washed and stained with Streptavidin Phycoerythrin (Molecular Probes) in Affymetrix fluidic stations, then detected by fluorometric scanning (Hewlett-Packard GeneArray Scanner). The quality of each microarray image is carefully examined for chip defects or abnormalities in hybridization signal prior to analysis with Affymetrix Microarray Suite (v. 5.0) and LIMS (v. 3.0). No data are entered into the database without passing more than 30 quality control metrics developed by Gene Logic.
For gene targets, the specificity of gene expression in human tissues is a common metric that is used to profile and qualify them as potential drug targets. ESR1 gene expression intensities were examined across a panel of 54 normal human tissues. Figure 1 shows a subset of the e-Northern report highlighting normal human tis