Nucleospin system

Deep-frozen tissue samples from primary and recurrent glioblastomas of 10 patients treated with TMZ/RT→TMZ after surgery were selected from the tumor tissue bank of the Department of Neuropathology, Heinrich Heine University, Düsseldorf, Germany, and investigated as approved by the institutional review board (study number 3825). Tissue samples were snap frozen immediately after resection and stored at −80°C. Each tissue specimen was histologically evaluated for sufficient tumor cell content. miRNA was extracted from frozen tissue samples or cell Qiagen, Hilden, Germany). Total RNA was prepared using the NucleoSpin System (Macherey-Nagel AG, Oensingen SO, Switzerland). RNA quantity was determined with the NanoDrop ND-1000 spectrophotometer (PeqLab, Erlangen, Germany). miRNA and mRNA quality assessment was done with an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). Only RNAs with a RNA integrity number of ≥ 7 were used for molecular analyses.

Total RNA was prepared with the NucleoSpin System (Macherey-Nagel, Düren, Germany) and cDNA was transcribed using Superscript II reverse transcriptase (Bio-rad, Munich, Germany). For real-time PCR, cDNA amplification was monitored using SYBR Green chemistry on the 7300 Real time PCR System (Applied Biosystems, Zug, Switzerland). The conditions for these PCR reactions were: 40 cycles, 95°C/15 sec, 60°C/1 min, using the primers specified in Supplementary Table 1. Arf1 transcript levels were used as a reference for relative quantification of mRNA expression levels using the ΔCT method because they were observed to be stable within each cell line under the experimental conditions reported here.