Thermo fisher cloud platform

Eight single-nucleotide polymorphisms (SNPs) (C1584G, C100T, C1023T, G1846A, C2580T, G2988A, G3183A, and G4180C) and one deletion (2615_2617delAAG) in the CYP2D6 gene and the copy number of the gene were assayed by qPCR. We used specific hydrolysis probes for each polymorphism (TaqMan SNP genotyping assays; Thermo Fisher Scientific). All amplification reactions were performed according to the protocol described by Silvino and colleagues (15 (link)). Amplification and fluorescence detection were carried out using the ViiA 7 real-time PCR system (Thermo Fisher Scientific). The results were analyzed by QuantStudio real-time PCR software (Thermo Fisher Scientific) and the Thermo Fisher cloud platform (Thermo Fisher Scientific).
The copy number of the CYP2D6 gene was determined by quantitative PCR (qPCR) using the Hs00010001_cn assay (Thermo Fisher Scientific) for gene deletion and amplification detection. All amplification reactions were performed according to the protocol described by Silvino and colleagues (15 (link)). Amplification and fluorescence detection were carried out in the ViiA 7 real-time PCR system (Thermo Fisher Scientific), and the number of copies was estimated in CopyCaller v.2.0 software. Only samples with confidence values greater than 95% and absolute z-scores of <1.75 were considered in our analysis.

The genotyping of A503V (rs1057868), which changes GCC to GTC at codon 1508 in the gene that encodes CPR, was assayed by qPCR. We used a specific hydrolysis probe for the target region (Assay ID C___8890131_30; Thermo Fisher Scientific). All amplification reactions were performed in 384-well plates in a total volume of 5 µL in the presence of 2.5 µL 2 × TaqMan universal PCR master mix (Thermo Fisher Scientific), 0.25 µL TaqMan SNP genotyping assay reagent (Thermo Fisher Scientific), 1.25 µL ultrapure water (free of nucleases), and 1 µL target DNA (≅ 10 ng/ µL). A negative control was prepared containing no template DNA, and heterozygous and homozygous samples were used as positive controls. The thermocycling conditions were initial denaturation at 95°C for 10 min, followed by 50 cycles of 15 sec at 92°C and 90 sec at 60°C, and one cycle of 30 sec at 60°C. Amplification and fluorescence detection were carried out using the ViiA 7 real-time PCR system (Thermo Fisher Scientific). The results were analyzed by QuantStudio real-time PCR software (Thermo Fisher Scientific) and the Thermo Fisher cloud platform (Thermo Fisher Scientific).