Invisorb blood universal kit

DNA isolation was performed using 7 ml aliquots of fresh or frozen whole blood (Fig 2; n = 192). Maxi DNA extraction kits from 5 different suppliers (GeneCatcher gDNA Kit from Invitrogen, QIAamp DNA Blood Maxi Kit from Qiagen, NucleoSpin Blood XL Kit from Macherey-Nagel, PerfectPure DNA Blood Kit from 5prime and Invisorb Blood Universal Kit from Stratec) as well as one published isopropanol precipitation protocol (IPP) [30 (link), 31 (link)] were used. DNA extraction kits used magnetic beads (Invitrogen), large (Marcherey-Nagel, Qiagen) and small (5prime) spin columns or precipitation (Stratec, IPP). DNA isolation was performed according to the manufacturers’ instructions and DNA was eluted in 300 μl (5prime kit), 600 μl (IPP), 1000 μl (Marchery-Nagel, Qiagen), 1400 μl (Stratec) or 1500 μl (Invitrogen). After elution, DNA was quantified using OD measurements performed in duplicates for each sample on a NanoDrop ND-1000 spectrophotometer (Fisher Scientific) and was stored at -20°C until further use. An overview about the characteristics of the kits can be found in Table 1. Before qPCRs, samples were diluted to a DNA concentration of 10 ng/μl and quantified with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) on a SpectraMax Paradigm Multi-Mode Microplate Detection Platform (Molecular Devices) before subsequent analyses. One sample was lost due to handling errors.

From each participant, we obtained a total of 5 ml EDTA-treated peripheral blood. Human nuclear DNA was isolated from PBMCs using an Invisorb Blood Universal Kit (Stratec Molecular GmbH; Berlin, Germany) following the manufacturer's specifications. DNA concentrations were spectrophotometrically measured based on the default OD 260/280 absorbance algorithm. Then the DNA was diluted to a concentration of 5 ng/μl and stored at −20°C until use.

DNA was isolated using the Invisorb^® Blood Universal Kit from Stratec Molecular (Berlin, Germany). Eleven well-known T2DM genetic risk variants were genotyped: TCF7L2(rs7903146), PPARG-P12A(rs1801282), DUSP9(rs594532), CENTD2(rs1552224), THADA(rs7578597), HHEX(rs1111875), CDKAL1(rs7754840) and KCNQ1(rs231362)which had previously been genotyped for replication in DIAGRAM [37 (link)], and KCNJ11(rs5219), IGF2BP2(rs4402960) and FTO(rs8050136). These risk variants were chosen because of their relatively large effect sizes on T2DM risk in previous studies [32 (link), 37 (link)–42 (link)]. Genotyping was performed with TaqMan allelic discrimination assays, designed and optimized by Applied Biosystems (Foster City, CA, USA). Reactions were performed on the Taqman Prism 7900 HT platform.