Flexigen 250 kit

Molecular analyses were carried out on genomic DNA extracted from iPSCs using the Flexigen 250 kit (Qiagen, Hilden, Germany). Beta S (β^S) globin gene cluster haplotypes were investigated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method [37 (link)]. Briefly, PCR amplification was performed using five primer pairs (3/4, 5/6, 6/7, 8/9, and 10/11). The reaction mixture contained genomic DNA, primer (25 pmol/μL), dNTPs (2 mM) (Thermo Fisher Scientific), Taq polymerase (5U/μL), MgCl₂ (50 mM), buffer 10X (Taq DNA Polymerase Kit, Thermo Fisher Scientific), and a sufficient volume of free DNase H₂O for 50 μL. PCR was performed at 94 °C for 10 min for initial denaturation, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at a specific temperature (Table 1) for 45 s and an extension at 72 °C for 90 s, and a final extension at 72 °C for 10 min. Amplicon (20 μL) was digested with the restriction enzymes XmnI, HincII, HindIII, or HinfI (Thermo Fisher Scientific). Buffer 10X and a suitable amount of PCR H₂O were added to this mixture. Regarding the reaction containing XmnI, bovine serum albumin was added. Amplicons and digested DNA fragments were electrophoresed on 1% and 2% agarose gels (Thermo Fisher Scientific) for 1–2 h, respectively, and visualized under ultraviolet light (Table 1).

Molecular analyses were carried out on genomic DNA extracted from peripheral blood leukocytes using the Flexigen 250 kit (Qiagen, Hilden, Germany) following the manufacturer's guidelines. Specific primers were used to identify R192Q and M55L mutations to PON1.
Primers of position 192 were as follows: 5′TAT.TGT.TGC.TGT.GGG.ACC.TGA.G3′ and 5′CAC.GCT.AAA.CCC.AAA.TAC.ATC.TC3′ for 99 bp DNA; of position 55 were 5′GAA.GAG.TGA.TGT.ATA.GCC.CCA.G3′ and 5′TTT.AAT.CCA.GAG.CTA.ATG.AAA.GCC3′ for 170 bp. The PCR products were digested with AlwI to R192Q and CviAII to M55L.