Gene expression profiling analysis

After RNA isolation, purity and integrity were measured using the NanoDrop ND1000 spectrophotometer and the Agilent Bioanalyzer electrophoresis system, respectively. High quality RNA was then used for the synthesis of biotin-labeled cRNA using the Illumina RNA amplification kit (Ambion) as described previously [57]. Briefly, 500 ng total RNA was converted to cRNA by in vitro transcription, and then purified 1.5 μg cRNA was hybridized to Illumina HT12 v4 (Illumina) chip. The slides from Direct Hybridization (Illumina) were then washed and scanned with the Bead Station 500 System (Illumina). The signal intensities from the scanner were quantified using GenomeStudio Software (Illumina). Quantile normalization in linear models was used to normalize the data.

Whole genome mRNA expression profiling was performed on primary tumors, lymph nodes, metastases and CTCs. Expression data are available at NCBI GEO ({"type":"entrez-geo","attrs":{"text":"GSE48496","term_id":"48496"}}GSE48496). A class comparison tool within BRB ArrayTools (National Cancer Institute, version 4) was used to select genes that are differentially expressed between the different tissue sites (6 replicates for each group). The values were averaged over replicates of samples and a two-sample t test was used to calculate the significance of the observations (FDR < 0.05 and p < 0.001). WebGestalt (http://genereg.ornl.gov/webgestalt) was used to perform KEGG pathway analyses [58]. Finally, to visualize expression patterns of specific genes of interest, expression values of these genes were adjusted to a median of zero and then analyzed with Cluster and TreeView [59].