Agilent human 44k 60 mer oligonucleotide microarray chips

Tissue samples from wide excision of the primary tumor were collected, placed in RNA later (Qiagen, Hilden, Germany), and immediately preserved in liquid nitrogen. The specimens were securely de-identified through a centralized database. All samples were confirmed to contain >95% tumor cells. RNA was extracted from the frozen tissue samples using the Qiagen RNeasy Mini Kit. The RNA concentration was measured by NanoDrop Spectrophotometer (NanoDrop Products, Wilmington, DE) and the quality determined by the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). All RNA samples were processed for microarray assay according to the manufacturer’s protocol. Briefly, RNA was labeled using the Quick AMP Labeling Kit (Agilent Technologies), purified on RNeasy columns (Qiagen), and hybridized on Agilent Human 44K 60-mer oligonucleotide microarray chips (Agilent Technologies, Santa Clara, CA). Microarray chips were incubated at 60°C for 16–17 h, washed, covered with ozone barrier, and then run through the Agilent Scanner (G2565CA). Agilent Feature Extraction Software (version 9) was used to quantify the intensity of fluorescent images.