normalization data transformation protocol
Data were normalized using lowess and mixed models where dye and array were treated as fixed and random effects, respectively, then log2 transformed
array scanning and feature extraction protocol
Slide images were captured using the Packard Bioscience ScanArray Express (PerkinElmer Inc., Waltham, MA), and spot intensities were assayed for each channel using ImaGene software (Biodiscovery Inc., El Segundo, CA)
nucleic acid hybridization to array protocol
Samples were competitively hybridized to Agilent custom oligonucleotide microarrays (Earray design ID 033450) and incubated at 60 degrees C for 18 hrs in a SureHyb microarray hybridization chamber (Agilent Technologies, Santa Clara, CA).
nucleic acid labeling protocol
Samples were coupled with Alexa fluor dyes (Alexa fluor 555 and 647; Molecular Probes, Inc.)
nucleic acid extraction protocol
Gill RNA (following tissue preservation in RNA later) was extracted following tissue homogenization in Trizol reagent (Invitrogen Inc.) and spin column purification.
Gill tissues were immediately dissected from adult animals and stored in RNAlater
We compared trajectories of gene expression change through time among populations and species using mixed model analysis of variance in JMP Genomics, where main effects were “species” and “time”, and interactions were specified. Five biological replicate fish were included per treatment; replicates were not pooled. “Dye” was treated as a fixed effect, “array” was treated as a random effect, and replicate individuals within treatment (time-by-species) was treated as a random effect.