*2.8. MDM2 and CDKN2A Gene Copy Number Analysis*

Genomic DNA was extracted using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) and gene amplification was evaluated by real-time PCR. Reactions were carried out using the following primers: for MDM2 gene, Fw 5- -TGGCTGTGTTCAAGTGGTTC-3 and Rv 5- -GTGGTGACAGGGTGCTCTAAC-3- ; for CDKN2A gene, Fw 5- -CACATTCATGTGGGCATTTC-3 and Rv 5- -TGCTTGTCATGAAGTCGACAG-3- (Exon 3, recognizing both p14ARF and p16INK4 sequences); and for the reference gene RPPH1 (ribonuclease P RNA component H1), Fw 5- -GAGGGAAGCTCATCAGTGG-3 and Rv 5- -ACATGGGAGTGGAGTGACAG-3- . Dissociation curve analysis of all PCR products showed a single sharp peak and the relative gene copy number was calculated using the 2−ΔΔCT method. For each set of samples, DNA from the corresponding healthy tissue was used as a calibrator.
