Identi®cation and removal of contaminating ̄uorescence from commercial and in-house printed DNA microarrays

Microarray analysis is a critically important technology for genome-enabled biology, therefore it is essential that the data obtained be reliable. Current software and normalization techniques for microarray analysis rely on the assumption that ̄uorescent background within spots is essentially the same throughout the glass slide and can be measured by ̄uorescence surrounding the spots. This assumption is not valid if background ̄uorescence is spot-localized. Inaccurate estimates of background ̄uorescence under the spot create a source of error, especially for low expressed genes. We have identi®ed spot-localized, contaminating ̄uorescence in the Cy3 channel on several commercial and in-house printed microarray slides. We determined through mock hybridizations (without labeled target) that pre-hybridization scans could not be used to predict the contribution of this contaminating ̄uorescence after hybridization because the change in spot-to-spot ̄uorescence after hybridization was too variable. Two solutions to this problem were identi®ed. First, allowing 4 h of exposure to air prior to printing on to Corning UltraGAPS slides signi®cantly reduced contaminating ̄uorescence intensities to approximately the value of the surrounding glass. Alternatively, application of a novel, hyperspectral imaging scanner and multivariate curve resolution algorithms, allowed the spectral contributions of Cy3 signal, glass, and contaminating ̄uorescence to be distinguished and quanti®ed after hybridization.