The labeled DIG-cRNA (10 μg per microarray) was injected into each microarray hybridization chamber. Following hybridization at 55C for 16 hours, the unbound material was washed from the microarrays. Features that retained bound DIG-labeled cRNA were visualized using the Applied Biosystems Chemiluminescence Detection Kit. The kit uses anti-DIG alkaline phoshatase that hydrolyses a chemiluminescent substrate to generate light at 458 nm which can be analyzed using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer.
Technical and Biological Replicates The reproducibility of the Applied Biosystems Expression Array System was demonstrated by comparison of technical replicates. Global median normalization was performed for each microarray and subsequent values were then log transformed. To assess variation in gene expression, background noise was filtered out for each pair of arrays by using only datawith signal-to-noise ratios greater than 3 (Figure 2A, Table 1). Additionally, we compared biological replicates (Biological_rep1 vs. Biological_rep2) between two individuals with the same disease condition (Figure 2B).
Fold Changes of Differential Gene Expression The fold change of normal vs. metastasis tumor sample was analyzed by filtering the dataset using p-values < 0.01 and a signal-to-noise ratio > 3 for use in ANOVA statistical analysis. Of these 2,508 genes, 623 genes were up-regulated and 435 genes were down-regulated. The number of detected genes for the corresponding p-values of < 0.05 and < 0.001 were 5,703 and 796 genes, respectively (Table 2). Using the Spotfire software application, fold changes can be determined for the differentially expressed genes (Figure 3).
From the ANOVA statistical analysis (p < 0.01) the identified 2,508 genes were sele