Taqman genotyping protocol

DNA was extracted using QIAamp DNA Blood Mini Kits-50-Catalog no. 51104 supplied by QIAGEN. DNA integrity was determined by 1% agarose gel electrophoresis, stained with ethidium bromide, and visualized through GEL documentation (E-Gel® Imager System with UV Light Base, Thermo scientific). DNA concentration was determined by NanoDrop 2000 Spectrophotometer (Thermo scientific). PPAR-γ Pro12Ala polymorphisms (rs1801282) were detected by Real-Time polymerase chain reactions (PCR) using the Quantistudio 12 Flex real-time PCR system (Applied Biosystems, CA 94404, USA). PPAR-γ Pro12Ala polymorphisms (rs1801282): (Applied BiosystemsID: C_1129864_10) allele discrimination was performed using the TaqMan® genotyping protocol (Applied Biosystems, Foster City, CA, USA). PCR reactions were set up in 20 μl reaction volume including 20–30 ng DNA and 10 μl TaqMan® Universal PCR Master Mix in 96-well PCR plates. The PCR assay was carried out according to manufacturer's instructions including one step of 10 min at 95 °C followed by 40 cycles of DNA denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Final products were analyzed by TaqMan Genotyper software.