Genechip scanner 3000 g7

Total RNA was isolated and reverse transcribed for hybridization to the Mouse Gene 1.0 ST Array (GeneChip; Affymetrix, Santa Clara, CA, USA) as described previously.^{33 (link)} Arrays were processed using the Affymetrix GeneChip Fluidic Station 450 (protocol EukGE-WS2v5_450) and scanned using a GeneChip Scanner 3000 G7 (Affymetrix). GeneChip Operating Software (Affymetrix GCOS v.1.4) was used to obtain chip images, with quality control performed using the AffyQCReport software (Bioconductor, Berkeley, CA, USA). GSEA was performed on the Board Institute Platform^{34 (link), 35} and statistical significance (false discovery rate) was set at 0.25. Microarray data are available from Gene Expression Omnibus (National Center for Biotechnology Information, Bethesda, MD, USA) under the accession number GSE45232.

Total RNA were reverse transcribed and used for in vitro transcription to generate labeled cDNA using Affymetrix 3' IVT Express Kit (Santa Clara, CA, USA) following manufacturer protocol and recommendations. Affymetrix HG-U133A v2.0 GeneChip microarrays for gene expression (n = 31) were hybridized and scanned on an Affymetrix GeneChip Scanner 3000 G7. Quality control and normalization were performed as reported previously.[17 (link)] Probe sets raw intensities were stored in electronic files (.DAT and .CEL formats) by the GeneChip Operating Software (GCOS). Statistical analyses were performed over all probesets (n = 22,277) on each GeneChip microarray including control probesets to discard significant differences. A two-sample t-test was fit for TAC vs. EVR comparison in the R programming environment.[18 ] A p-value <0.05 was considered significant for differentially expressed genes. Differential gene expression was illustrated using fold-changes.

Total RNA was purified from xenograft using TRIzol^® Reagent (Gibco, Life Technologies) according to the manufacturer. Briefly, 50–100 mg of fresh frozen tissue per ml of TRIzol^® was disrupted using a homogenizer followed by a single step of phenol/chloroform purification. Total RNA was quantified using the Nanodrop spectrophotometer (NanoDrop Technologies, Inc), and RNA Integrity Number (RIN) was calculated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA samples that reached a RIN between 8 and 10 were used for microarray hybridization (GeneChip; Affymetrix Inc., Santa Clara, CA). The Genechip^® Human Gene 2.0 ST Arrays were washed and stained using the Affymetrix GeneChip fluidic station 450 (protocol EukGE‐WS2v5_450) and were scanned using a GeneChip scanner 3000G7 (Affymetrix Inc., Santa Clara, CA). GeneChip operating software version 1.4 (Affymetrix Inc., Santa Clara, CA) was used to obtain chip images and for quality control. Background subtraction and normalization of probe set intensities were performed using the method of Robust Multi‐array Analysis (RMA; Irizarry et al, 2003). Microarray analysis was performed by the CHU de Québec Research Center Gene Expression Platform (Quebec City, Quebec, Canada). Seventeen samples of the cohort were previously published (Duconseil et al, 2015) as GEO accession numbers GSE55513 and GSE89792.