DNA Extraction and Genotyping

Genomic DNA was extracted from 3 to 4 (5-μm-thick) sequential sections for each of the FFPE samples after deparaffinization.³⁰ Wax was removed from the specimens by adding 500-μL heptane to a 2-mL microcentrifuge tube containing 3 to 4 sections of paraffin-embedded tissue and incubation at room temperature for 10 minutes to dissolve the wax. Then 25 μl of Methanol (100%) were added, and the tube was vortexed for 10 seconds. The supernatant was removed after centrifugation at 12 000-16 000 rpm in a microcentrifuge for 2 minutes. One milliliter of 96% ethanol was added, and the tube was vortexed for 10 seconds. After proteinase K treatment, DNA was extracted from the resulting pellet with the commercially available AllPrep DNA/RNA FFPE kit (Qiagen, Hilden, Germany), using the manufacturer's protocols. The quality of extracted DNA was examined by agarose gel electrophoresis and ethidium bromide staining. Extracted DNA was quantified using the PicoGreen dsDNA Assay kit (Invitrogen, Carlsbad, CA) and normalized to a concentration of 50 ng/μL. Genotyping on Illumina Chip HumanOmniExpress-24 v1.0 arrays (Illumina Inc., San Diego, CA) was performed according to the manufacturer's instructions and as reported previously.³¹