Microarray analysis
Normalisation of the expression level was done using Affymetrix software MAS 5, which is based on global scaling of total gene expression level per microarray. The normalised expression values were imported to and analysed by dCHIP [24]. Differentially expressed genes were identified by defining the appropriate filtering criteria in the dCHIP software as: lower 90% confidence boundary of fold change between the group means exceeded twofold; the absolute difference between the two groups exceeded 100; the P value threshold of the unpaired t-test was 0.05. The false discovery rate was established with a permutation test for each pairwise comparison to estimate the proportion of false-positive genes.
Hierarchical gene clustering was performed with dCHIP to characterise the gene expression patterns during CIA. The default clustering algorithm of genes was as follows: the distance between two genes is defined as 1 – r, where r is the Pearson correlation coefficient between the standardised expression values of the two genes across the samples used. To characterise the functional relationship between differentially expressed genes, Gene Ontology (GO) term classification incorporated in DNA-Chip Analyzer was performed. The significant level for a function cluster was set at P < 0.005, and the minimum size of a cluster was three genes.