Seventy-six fresh-frozen surgical samples of de novo GBMs were prospectively collected between 2010 and 2015. RNA was extracted with the Nucleospin® TriPrep (Macherey-Nagel, Düren, Germany) and the QIASymphony RNA (Qiagen, Venlo, The Netherlands) kits according to the manufacturers’ instructions. Affymetrix HG U133 plus 2.0 arrays were prepared and scanned according to the manufacturer’s protocol and as reported previously [54 (link)]. Quality control and differential gene expression analyses were performed with R (v3.2.2). Based on principal component analysis (PCA) plots, 3 outliers were removed. Robust Multi-array Average (RMA) normalization was applied. Batch correction was performed with the ‘sva’ package. Differential expression was analyzed with the ‘limma’ package and heatmaps were created with the ‘heatmap3′ package (R).
Exploratory Gene Set Enrichment Analyses (GSEA) were performed after RMA-normalization [55 (link)] and batch correction, with the Partek® Genomics suite platform (v6.6) (Partek, St. Louis, MO, USA). Analyses were performed with the Broad Institute MySig libraries of curated gene sets C1—C7 version 5.0 [56 (link)], 1000 permutations and default additional parameters. An FDR threshold of 0.25 was applied as recommended [55 (link)].
Exploratory Gene Set Enrichment Analyses (GSEA) were performed after RMA-normalization [55 (link)] and batch correction, with the Partek® Genomics suite platform (v6.6) (Partek, St. Louis, MO, USA). Analyses were performed with the Broad Institute MySig libraries of curated gene sets C1—C7 version 5.0 [56 (link)], 1000 permutations and default additional parameters. An FDR threshold of 0.25 was applied as recommended [55 (link)].
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