Microarray analysis was performed as described previously [25]. Briefly, fluorescence-labeled RNA targets were prepared from 5 µg of total RNA using MessageAmp™ aRNA kit (Ambion, Austin, TX, USA) and Cy5 dye (Amersham Pharmacia, Piscataway, NJ, USA). Fluorescent targets were hybridized to the Mouse Whole Genome OneArray™ (Phalanx Biotech Group, Hsinchu, Taiwan) and scanned by an Axon 4000 scanner (Molecular Devices, Sunnyvale, CA, USA). Three replicates from three independent mice were performed. The Cy5 fluorescent intensity of each spot was analyzed by genepix 4.1 software (Molecular Devices). The signal intensity of each spot was corrected by subtracting background signals in the surrounding. We filtered out spots that signal-to-noise ratio was less than 0 or control probes. Spots that passed these criteria were normalized by the R program in the limma package (http://www.r-project.org/). Genes with fold changes ≥2.0 or ≤−2.0 were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (http://www.genome.ad.jp/kegg/), which is a knowledge base linking a set of genes with a network of interacting molecules in the cells [27]. We used the WebGestalt tool to test significant KEGG pathways.
