Figure 3

Normalization of a two-channel hybridization. A The linlog transformed data before normalization plotted as channel 1 (Alexa 546 dye) versus channel 2 (Alexa 647 dye) intensities. B,C Ratio-Intensity plot before (B) and after (C) spatial and intensity lowess normalization. "M" refers to the log ratio of the two channels and "A" refers to the geometric mean of the spot intensity for both channels. The blue line in B is the spatial/intensity lowess normalization fitted curve, with the curvature of the line representing the intensity-dependent fit and the width of the line representing the spatial component of the lowess normalization. D The final normalized data used for the ANOVA. E A plot of the correction factors, by array position, used to normalize intensities due to spatial effects only. Shown are the amount adjusted for each of the 650 elements in the 12 × 4 subarrays (in the X and Y directions, respectively) of the mouse cDNA microarray used in the study. A location depended effect can be observed as a general increase over the length of the slide (bottom to top) and a dip near the center of the slide.