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Microarray analysis is a critically important technol- ogy for genome-enabled biology, therefore it is essential that the data obtained be reliable. Current software and normalization techniques for micro- array analysis rely on the assumption that fluores- cent background within spots is essentially the same throughout the glass slide and can be meas- ured by fluorescence surrounding the spots. This assumption is not valid if background fluorescence is spot-localized. Inaccurate estimates of back- ground fluorescence under the spot create a source of error, especially for low expressed genes. We have identified spot-localized, contaminating fluor- escence in the Cy3 channel on several commercial and in-house printed microarray slides. We deter- mined through mock hybridizations (without labeled target) that pre-hybridization scans could not be used to predict the contribution of this contaminat- ing fluorescence after hybridization because the change in spot-to-spot fluorescence after hybridiza- tion was too variable. Two solutions to this problem were identified. First, allowing 4 h of exposure to air prior to printing on to Corning UltraGAPS slides significantly reduced contaminating fluorescence intensities to approximately the value of the sur- rounding glass. Alternatively, application of a novel, hyperspectral imaging scanner and multivariate curve resolution algorithms, allowed the spectral contributions of Cy3 signal, glass, and contaminat- ing fluorescence to be distinguished and quantified after hybridization.

Identification and removal of contaminating fluorescence from commercial and in-house printed DNA microarrays