A variety of methods have been used to analyze data obtained from protein microarrays [22, 25, 27-31]. These methods have been primarily adopted from those used in gene microarray analysis. The analysis of RPAs presents a new set of challenges compared with conventional spotted arrays. Multiple RPAs, each analyzing a different phosphorylated protein, are scanned; the background is subtracted; spot intensities are calculated and normalized; and the dilution curve is collapsed to a single intensity value measured within the linear dynamic range of the reference standard. This value is then assigned a relative normalized intensity value referenced to the other patient samples on the array. The data output is in a form suitable for analysis by traditional unsupervised and supervised bioinformatics computer systems. Ultimately, protein array data are displayed as traditional "heat maps," which can be analyzed by Bayesian clustering methods for signal-pathway profiling (Figure 7.4).
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