Mx3005p platform

DNA was isolated from 200 μL EDTA‐anticoagulated blood with a DNA extraction kit (Epicenter Kit, Nordic Biolabs, Sweden) and kept at −20°C. Three selected SNPs in the SERT gene with possible functional impact and association with MMVD were: c.814insG (p.Lys272Arg), c.1193delT (p.Val397Gly), c.1324G>A (p.Gly442Ser; Table 1).¹⁹ Single nucleotide polymorphism genotyping was performed by TaqMan assays established based on sequence information extracted from Ensembl, comprising 100 to 200 bp sequences on both sides of the polymorphisms. Primers and probes were designed for wild type and mutated sequences, respectively, by Thermo Fischer Scientific, Denmark using VIC for wild type alleles and FAM for variant alleles. TaqMan real‐time PCR was carried out as follows on the Mx3005P platform (Agilent, Glostrup, Denmark): 1 μL of genomic DNA (20‐25 ng/μL) was amplified in a total volume of 10 μL reaction mastermix containing 5.0 μL TaqMan Universal Mix, 0.25 μL TaqMan enzyme (Thermo Fischer Scientific, Denmark) and 3.75 μL H₂O. PCR conditions were: 1 cycle of 50°C for 2 minutes, 1 cycle of 95°C for 10 minutes, and 45 cycles of 92°C for 20 seconds followed by 60°C for 1 minute. Water was included as negative control and results were analyzed by MxPro qPCR Software (Agilent, Glostrup, Denmark).