Qiaamp mini spin columns kit

The DNA was extracted from whole blood samples (ethylenediaminetetraacetic acid) using the QIAamp Mini Spin Columns kit (QIAGEN, Venlo, the Netherlands) in accordance with the manufacturer’s instructions. For detection of polymorphic variable, the real-time polymerase chain reaction method in the TAQMAN® (Thermo Fisher Scientific, Waltham, MA, USA) system was used.
We genotyped the following SNP of the VDR gene: FokI (rs10735810/ID: C__12060045_20) and TaqI (rs731236/ID:C___2404008_10) according to the manufacturer’s instructions (http://www.thermofisher.com/order/genome-database). FokI polymorphism results in different translation initiation sites due to the replacement of cytosine (C) by thymine (T) and TaqI polymorphism is constituted by the silent replacement of thymine (T) by cytosine (C).

The biological material (blood) was collected by vacuum venipuncture for DNA extraction with a commercial kit. The DNA samples were extracted from whole blood (EDTA) with QIAamp Mini Spin Columns kit (Qiagen) following the instructions from the manufacturer. In the next step, the DNA samples were stored at −20ºC until the time during which the genotyping assays were performed. Genotyping methodology was used in real-time PCR, through the TAQMAN® system by using fluorescent probe CYP2C9*2 (rs1799853) assay (C__25625805_10), CYP2C9*3 (rs1057910) assay (C_27104892_10) and VKORC1 -1639G>A (rs9923231) assay (C_30403261_20). Reactions were carried out using TaqMan Genotyping mastermix (Thermo Fisher Scientific, MA, USA) according to the instructions from the manufacturer. For genotyping, the 5 QuantiStudio equipment (Thermo Fisher Scientific, MA, USA) was used. All reactions were performed at the Instituto Aggeu Magalhães-FIOCRUZ-PE.