Taqman genotyping assay

Eight SNPs in the TGFBR3 gene were selected according to NCBI SNPs database: rs883873, rs2770186, rs12141128, rs12566180, rs6680463, rs1805110, rs1805113, rs2296621. All SNPs were supposed to have minor allele frequency (MAF) ≥5% and localization assigned as 5′ regulatory region, intron or exon of the TGFBR3 gene, which is located on chromosome 1. Characteristics of studied SNPs are presented in Table 2.
Real-Time PCR method with TaqMan Genotyping Assays (Thermo Fisher Scientific, Waltham, MA, USA) was applied for SNPs genotyping. The characteristics and sequences of used TaqMan probes are shown in Supplementary Table S1. PCR amplifications were conducted in a total volume of 10 µL and consisted of 5 µL (2×) of TaqMan Genotyping Master Mix buffer (Thermo Fisher Scientific, Waltham, MA, USA), 0.25 µL (40×) TaqMan Genotyping Assay (Thermo Fisher Scientific, Waltham, MA, USA) and 10 ng of template DNA. Thermal conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of sequential incubations at 95 °C for 15 s and at 60 °C for 1 min and final endpoint measurement of fluorescence. Real-Time PCR amplifications and allelic discrimination were performed using Mastercycler^®ep realplex (Eppendorf, Hamburg, Germany).

Deoxyribonucleic acid (DNA) extraction and genotyping of selected single nucleotide polymorphisms (SNPs), CFH (rs1061170, rs1410996) and KDR (rs2071559, rs1870377), were conducted at the Laboratory of Ophthalmology, Neuroscience Institute, Lithuanian University of Health Sciences. Utilizing predesigned TaqManTM genotyping assays from Thermo Fisher Scientific, Pleasanton, CA, USA, the procedures were executed in accordance with the manufacturer’s instructions, following established protocols. To ensure high-quality DNA samples, the DNA salting-out method was selected for DNA extraction.
SNPs were determined using TaqMan^® genotyping assays (Applied Biosystems, New York, NY, USA; Thermo Fisher Scientific, Inc., Waltham, MA, USA), C___8355565_10, C___2530294_10, C__15869271_10 and C__11895315_20 according to manufacturer’s protocols by a StepOne Plus (Applied Biosystems, Waltham, MA, USA). For each reaction, 1 μL of genomic test DNA and 9 μL of PCR reaction mix were used. The composition of the PCR mixture and the PCR reaction conditions are given in Table 2 and Table 3, respectively.

The amplification procedures was applied to the − 634G/C polymorphism located in the promoter region of the VEGFA gene. The used primers and probes sequences were

VEGFA-5′-GAGAGAAGTCGAGGAAGAGAGAGA-3′ and

5′-CCCAAAAGCAGGTCA CTCACTT-3′

VEGFA-FAM-5′- CCTGTCCCTTTCGC-3′ and 5′- CCTGTCGCTTTCGC-3′.

The used kit is TaqMan Genotyping Assay (Life Technologies – Applied Biosystems – ABI, Foster City, CA). The real time amplification procedures were performed with total volume 5 µL containing 2 ng genomic DNA, TaqMan Genotyping Master Mix 1× (ABI), and Custom TaqMan Genotyping Assay 1× (ABI). PCR was performed in a 7500 fast real time PCR system (ABI). The amplification procedures included an initial cycle of 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min [23 (link)].

Genomic DNA was isolated from venous blood samples, according to the manufacturer’s protocol (Gentra Puregene Blood Kit, Qiagen, Germany). Genotypes of the Pro12Ala (rs1801282) and Trp64Arg (rs4994) polymorphisms were determined by a TaqMan genotyping assay (Life Technologies, Carlsbad, California, USA). As a quality control measure, negative controls and approximately 5% of samples were genotyped in duplicate to check genotyping accuracy. The controls for each of the genotypes of the both SNPs were run in parallel. An allelic discrimination assay was performed on an ABI7900HT or on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA). The genotypes were determined as Trp64Trp, Trp64Arg, and Arg64Arg without prior knowledge of the subjects’ status.