Quantitative Gene Expression Analysis

Gene expression was measured as already described [15 (link)]. Briefly, tumor samples, with more than 80% of tumor cells, were homogenized in RNA lysis buffer in ice with an Ultra-Turrax (IKA, Staufen, Germany), and RNA was purified using the Maxwell 16 LEV SimplyRNA Cells kit (Promega, Madison, WI, USA). In all the samples by real time-PCR, the % of murine DNA contamination was established using primers specifically designed to distinguish human from murine actin and only samples with more than 85% human DNA were processed. Retro-transcription to cDNA was done using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). Genes selected were DNA pol β, ERCC1 and XPF. Optimal primer pairs (Table S3) were chosen, spanning splice junctions, using Primer3 Input software (Primer3 Input. http://primer3.ut.ee/) and the human specificity was verified by detecting single-band amplicons of the PCR products. Absolute copy numbers of mRNA were determined by real time-PCR (ABI-7900, Applied Biosystems, Foster City, CA, USA) with the SYBR Green technique (Promega), using an EP Motion 5075 robot (Eppendorf, Hamburg, Germany). Standard curves for each gene were included for absolute quantification of mRNA.
Real time-PCR data were normalized using the geometric mean of two housekeeping genes, actin (ACTB) and ciclophillin (CYPA).