Fig. 2. Replicate hybridizations and reciprocal dye labeling experiments. (A) Duplicate hybridizations were performed for each time point in the first two temperature cycles. Expression data were filtered by accepting only spots that changed over twofold in at least 1 time point. These data are not corrected relative to t=0, but they are median centered. Cy3 was used to label the reference sample while Cy5 was used to label the experimental sample in the forward (F) hybridizations. The dyes were reversed (R) in the second set of hybridizations. Visual inspection reveals a strong relationship between the two sets of hybridizations, especially for spots that change greater than twofold compared to the reference. (B) There is a strong correlation between the data for the forward and reverse hybridizations (r=0.96) for spots on the array that change more than twofold (N=831). The equation for the regression line on the graph is y=1.061x+0.04056. These data indicate that there is no significant dye bias in this data set. Further, it supports the conclusion that changes in gene expression observed in this study are not likely to be due to variation in hybridizations, but instead are biologically relevant.