TMPRSS6 Genotyping by TaqMan Assay

Genomic DNA was extracted from 3 ml whole blood on EDTA by a commercial DNA extraction kit according to manufacturer’s protocol (QIAamp DNA Blood Mini kit, QIAGEN, USA) CAT No.51104. 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 Nano Drop 2000 Spectrophotometer (Thermo scientific). TMPRSS6 genotyping polymorphisms

rs4820268: CCTACCTTCCTGGCACTGCTCTTC [A/G] TCGCTGCCGTTGAGACAATCAGGCT, rs855791: GCGTGGCGTCACCTGGTAGCGATAG [A/G] CCTCGCTGCACAGGTCCTGTGGGAT rs11704654: CCTCACAGGCCTTGAACATCCCCTC[C/T] GGCTCCGCTTCCTCGCCATCACCTC

were 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, 10 μl TaqMan genotyping PCR Master Mix and 1 μl TaqMan SNP genotyping assay. 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 using the Rotor Gene Q real-time PCR (QIAGEN, Germany). Final products were analyzed by Rotor Gene software (Shinta et al.,) [22 (link)].