244k array

The second GBM dataset analyzed for the present study is available as supplementary material of a recent study[27 (link)]. Data include microarray expression profiles of 173 core TCGA samples unified and scaled from three gene expression platforms (Affymetrix HuEx array, Affymetrix U133A array, and Agilent 244K array). The authors of this study described a robust gene expression-based molecular classification of GBM into Proneural, Neural, Classical, and Mesenchymal subtypes and integrate multidimensional genomic data to establish patterns of somatic mutations and DNA copy number. Aberrations and gene expression of EGFR, NF1, PDGFRA/IDH1, and neuron markers (e.g. NEFL, GABRA1, SYT1, SLC12A5) each define the Classical, Mesenchymal, Proneural and Neural subtypes, respectively.

Level 3 mRNA expression (Agilent 244k array) and clinical parameters were downloaded from The Cancer Genome Atlas (TCGA) breast carcinoma dataset on October 18, 2013. A second set of previously published gene expression data for 129 breast tumors profiled using Affymetrix U133p2.0 microarrays were downloaded from the Gene Expression Omnibus (GSE5460). PhosphoSTAT3 immunohistochemistry for the corresponding tissue microarrays were previously reported [44 ]. Correlation analysis between the mRNA levels of GRN and genes in STAT3 expression signatures were conducted with GraphPad Prism 6 Software (La Jolla, CA). The significance of the correlation was calculated using Pearson's correlation. Kaplan-Meier survival analyses were performed with GraphPad Prism 6 Software. Differences in observed survival between groups were tested for significance using the log-rank (Mantel-Cox) test.

Genomic DNA was isolated from xenografts before the serial transplants (P0), after the first (P1) and the fifth transplantations (P5), using DNeasy extraction kit (Qiagen, France) according to the manufacturer's instructions. The percentage of mouse and human component was determined by quantitative PCR (qPCR) using species-specific primers. CGH labeling and hybridization were performed using high-density 244K arrays (Agilent, France). Sample DNAs were labeled with Cy5-dUTP and Cy3-dUTP, respectively. Labeled products were purified with Microcon YM-30 filters (Millipore, Billerica, MA). Arrays were scanned with an Agilent DNA Microarray Scanner (G2565BA). Log2 ratios were determined with Agilent Feature Extraction software (v9.1.3.1). The global quality of the individual microarrays was validated against the quality metrics (QCmetrics) provided in this software. Results were analyzed with Agilent's CGH Analytics v3.5 software. Copy number aberrations (CNA) were detected using the Aberration Detection Method algorithm 2 (ADM-2) with a threshold of 6. At least two contiguous suprathreshold probes were required to define a chromosomal abnormality. Percent of chromosomal abnormalities was defined as the number of CNA upon total number of probes.