Analyses of the quality and integrity of the extracted DNA

To verify the quality and integrity of the extracted DNA a multiplex PCR reaction with four pairs of primers for the GAPDH gene was performed as described by Van Beers et al. [36].

Basically, the PCR was performed with a final volume of 30 μL, containing 1.5 mM MgCl2; 0.2 mM dNTP (Invitrogen™); 0,133 μM of each primer; 1 U Taq DNA polymerase (Invitrogen™) and 60 ng normal/tumor DNA. The reactions were performed in a Veriti^® thermocycler (Applied Biosystems) using the following amplification parameters: 94°C for 1 minute, 35 cycles of 94°C for 1 minute, 56°C for 1 minute, and 72°C for 3 minutes. Finally, a final extension at 72°C for 7 minutes, finishing at 15°C. The successful amplification of DNA was checked by agarose gel electrophoresis on 1.5% GelRed ™ stained, visualized under UV light and documented.