Sample loading solution

Amplification products were separated and quantified according to the manufacturer’s instructions. Briefly, the PCR products were pre-diluted 20-fold with 10 mM Tris-HCl buffer pH 8.0. One μL of the diluted sample was separated with 39 μL Sample Loading Solution (Beckman Coulter) and 0.4 μL DNA size standard-400 (Beckman Coulter) on the GenomeLab Separation Capillary Array of the CEQ8800 (Beckman Coulter) using the Frag-3 separation program (denaturation for 120 s at 90 °C; sample injection for 30 s at 2.0 kV; separation of fragments for 40 min at 6.0 kV; capillary temperature at 50 °C). The raw data were analysed with the Fragment analysis module of the CEQ8800 software (Beckman Coulter) to estimate the size of the obtained fragments. The peak area of each fragment was exported for subsequent data processing.

Genomic DNA was extracted from macrodissected paraffin sections using the Maxwell® RSC FFPE Plus DNA Purification Kit and the Maxwell® 16 Instrument (Promega, Madison, WI), according to the manufacturer's instructions. Microsatellite PCR in duplicates was performed using genomic DNA and AmpliTaq Gold DNA Polymerase (ThermoFisher) as well-fluorescent labeled primers (Sigma-Aldrich). For GeneScan analysis PCR products were mixed with sample loading solution (Beckman Coulter). The products were separated by capillary electrophoresis on the GenomeLab GeXP Genetic Analysis System and analyzed by the GenomeLab GeXP software 10.2 (Beckman Coulter).