6000 nano chips

Microarray analyses were performed using RNAs obtained from three independent dishes of DLD-1/Mock and DLD-1/DKK-1 cells that were treated with doxycyclin (10 μg/ml) for 24 h. Total cellular RNA was isolated using the RNeasy kit (Qiagen) according to supplier's specifications. The quantity and quality of the total RNAs obtained were determined using 6000 Nano Chips (Agilent Technologies). Total RNA samples were then processed for hybridization on Gene Chip Human Gene 1.0 ST Array (Affymetrix) using standard Affymetrix protocols at the Genomics and Proteomics Facility of Centro de Investigación del Cáncer (Salamanca, Spain).
Analysis for differential expression was performed using the R platform for statistical analysis (R Foundation for Statistical Computing, Vienna) and several packages from the Bioconductor project (http://www.bioconductor.org/). The raw data were imported into R and preprocessed using the affy package and the robust multichip average method. To identify differentially expressed genes, we used the limma package. Correction for multiple testing was accomplished by controlling the FDR using published methods [58 ]. The experimental settings and raw data obtained in this experiment are deposited in GEO accession no. GSE35272.