Taqman real time pcr genotyping assays

The patients were genotyped for the following single-nucleotide polymorphisms (SNPs) within MT1A, MT2A and MT1L. The following variants were genotyped: MT1A rs11076161 A > G, MT2A rs28366003 A > G and MT1L rs10636 G > C. Genomic DNA was isolated from 0.2 mL of whole blood samples, using a commercial kit for genomic DNA isolation and the Genomic Mini AX Blood 1000 Spin (A&A Biotechnology, Gdańsk, Poland). TaqMan real-time PCR genotyping assays (Thermo Fisher Scientific) were used for the detection of the studied SNPs (Assay IDs: C_1402094_10, C_60284591_10, C_25996927_10).

Five SNPs in three critical ATG genes (ATG5 rs2245214 C >G rs510432 T >C; ATG10 rs1864182 A >C, rs1051423 T >C; ATG16L rs2241880 A>G) were selected from functional SNPs in the literature or that were associated with cancer or disease outcomes 15, 18, 19, 21, 27 (see Fig. 1). Five Taqman Real‐Time PCR Genotyping Assays (ThermoFisher Scientific, Grand Island, NY) were used to identify SNPs in ATG genes performed with a 7900HT Fast Real‐Time PCR System (ThermoFisher Scientific, Grand Island, NY) following manufacture recommendations. The ratio of fluorescence in amplification during the logarithmic phase was quantified to identify specific alleles in genes of interest using a commercially available Taqman primer assay on a 7900HT Applied Biosystems qPCR machine. The genotyping call rates ranged from 96% to 99%, and biological replicates were generated for 10% of the samples with 100% concordance.

For the study purpose, genomic DNA was isolated from 0.2 mL of a whole blood sample (all with blood cells) using a commercial kit for genomic DNA isolation using Genomic Mini AX Blood 1000 Spin (A&A Biotechnology). The genotyping of the selected SNPs was performed using pre-designed Genotyping Assays (TaqMan real-time PCR genotyping assays, Thermo Fisher Scientific, Assay IDs: C___2967992_1_, C___3237198_20). The following SNPs were genotyped: GSTP1 rs1695 A > G and SLC11A2 rs224589 T > G.