Hg u133 plus 2.0 genechip oligonucleotide arrays

After thawing, tumors were excised and lysed in Trizol Reagent (Life Technologies), according to the manufacturer' instructions. Briefly, tumors were homogenized (Dispomix, L&M Biotech, Holly Springs, NC, USA) and incubated in the Trizol solution for 2 minutes at room temperature, before the addition of chloroform. Tubes were vigorously shaken and the different phases separated by centrifugation. The upper, aqueous phase was recovered and the RNA present precipitated with isopropyl alcohol. Once washed in 70% ethanol the resultant RNA was column-purified (Qiagen, RNeasy mini kit) and its integrity was assessed (Agilent 2100 Bioanalyzer, Agilent). Biotinylated complementary RNA was then synthesized (Enzo Life Sciences) and hybridized to HG-U133 plus 2.0 GeneChip oligonucleotide arrays (Affymetrix). Quantitation of fluorescence intensities of probesets was done using the GenArray Scanner (Hewlett Packard). Unprocessed files were normalized using the RMA algorithm implemented in the Affimetrix Expression Console. Differentially expressed genes were identified using significant analysis of microarrays, selecting all genes with a value of Q≤0.05. Microarray data are now available through the GEO repository database (reference GSE75174).

We used HG U133 Plus 2.0 GeneChip oligonucleotide arrays (Affymetrix). Total RNA (8 μg) was used for synthesis of double-stranded cDNA. Biotinylated cRNA was synthesized with the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics). Both cDNA and cRNA were purified with GeneChip Sample Cleanup Module (Affymetrix). cRNA (16 μg) was fragmented and hybridized to the microarray for 16 h at 45 °C. The microarrays were stained, washed, and subsequently scanned with GeneChip Scanner 3000 (Affymetrix). Data were acquired using GCOS 1.2 software (Affymetrix). The preprocessing was performed by robust multi-array analysis (RMA, Bioconductor). Raw preprocessed data together with detailed descriptions of the samples are available at Gene Expression Omnibus repository under accession no Series GSE63885.

After assessment of RNA integrity (Agilent 2100 Bioanalyzer, Agilent), biotinylated complementary RNA was synthesized (Enzo) and hybridized to HG-U133 Plus 2.0 GeneChip oligonucleotide arrays (Affymetrix). Quantitation of fluorescence intensities of probesets was done using the GeneArray Scanner (Hewlett Packard). Microarray data have been deposited at GEO database (ref GSE46053).