RNA Isolation and cDNA Synthesis from Tumor Samples

RNA was isolated from 130 tumor samples before treatment using an RNeasy mini kit Plus containing DNase I (Qiagen, Germany) and Ribolock RNAse inhibitor (Thermo scientific, Waltham, MA, USA). The concentration and purity of RNA isolation were assessed with a NanoDrop-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA; 56–120 ng/μL; A₂₆₀/A₂₈₀ = 1.75–1.85; A₂₆₀/A₂₃₀ = 1.75–2.00). The RIN (RNA integrity number) was 6.4–7.9. The algorithm for assigning RNA integrity values was detailed by Schroeder A. et al. in 2006 [31 (link)]. To obtain cDNA on an RNA template, a reverse transcription reaction was performed using a RevertAid ™ kit (Thermo scientific, Waltham, MA, USA) with random hexanucleotides.