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The genes selected in this way were further filtered to remove those probe sets whose expression change under all experimental conditions was below a threshold, based on median normalized intensity values, which was considered to be the no-change threshold.
After selecting genes according to their P-value (limma; t-test) of significance (P <0.01), these genes were further filtered to remove those whose expression was below a threshold, based on median normalized intensity values, which was considered to be the no-change threshold (fold change >1.5).
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The spot intensities were median normalized (signal intensity of probe/median intensity of all discovery probes).
Technical replicates on arrays were collapsed by use of the median normalized relative intensity.
Genes were selected if the median normalized signal intensity was greater than 100 (75th percentile of median signal) for at least one group, with a mean change > 1.5-fold and a p-value < 0.05.
The protein intensity was calculated as the median of normalized intensity of the corresponding peptides.
A heat map of median normalized HITChip intensities and associated hierarchical clustering for all four samples was also produced using logarithmic Euclidean distances followed by complete-linkage clustering.
Data were median normalized in GeneSpring and normalized intensity data for each channel from each microarray were used to run GACK (Genomotypying Analysis Charlie Kim), to determine whether genes were present, absent, or divergent [ 56].
The average intensity of the 99 probes in this region in granitic plants is 1.2 (median normalized, where the median intensity = 1.0), while the serpentine plants have an average intensity of 2.6, which appears to indicate that there is one copy of this region in most granitic individuals and two or three copies in most serpentine individuals.
Background-corrected hybridization intensities were imported into BRB-ArrayTools, Version 4.2.1 [ 24] log2-transformed and median normalized; after normalization the distribution of signals remained uniform across all arrays.
Affymetrix probe set signal intensities were median normalized, i.e. divided by the median of each chip, and log10 transformed.
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