Tumor samples were snap frozen upon resection and stored at -80 oC until DNA extraction. Tumor content was assessed from equivalent material fixed in formalin and used for histological diagnosis (all samples contained ≥70% tumor cells).
Extracted molecule
total RNA
Extraction protocol
Total RNA from tumor specimens was isolated after milling of the frozen sample in a Micro-Dismembrator S (B. Braun, Melsungen, Germany), using TRIzol (Invitrogen, Carlsbad, USA) and subsequent purification with a RNeasy Mini Spin Column (Qiagen, Hilden, Germany). Integrity and quantitation of total extracted RNA was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE, USA).
Label
Biotin
Label protocol
Affymetrix protocol
Hybridization protocol
Affymetrix protocol
Scan protocol
Affymetrix protocol
Data processing
For normalization of the expression data we used the MAS5.0 algorithm of the GCOS program (Affymetrix Inc). Target intensity was set to 100 (α1= 0.04 and α2 = 0.06). Detection p-values were assigned to each probe set using the MAS5.0 algorithm (trimmed mean 96 = 100). Quality of the arrays was ensured by inspection of the ß-actin and GAPDH 5′-3′ ratios as well as the percentage of present calls generated by the MAS5.0 algorithm (Affymetrix Inc).
