Approximately 5 mL of blood collected from each participant was centrifuged to isolate leukocytes (3500 rpm, 20 °C, 30 min). Thereafter, a lysis buffer containing 3 μg/mL proteinase K was added to the cells. After extraction with TE-saturated phenol, ethanol precipitation was performed to purify the genomic DNA.
In total, 2 exons of ATP-binding cassette subfamily G member 2 (ABCG2) and 13 exons of solute carrier family 2 member 9 (SLC2A9/GLUT9 variants 1 and 2) were analyzed. To analyze two common SNPs of ABCG2 (rs72552713 and rs2231142), we performed TaqMan-based probe qualitative real-time PCR for genotyping using a THUNDERBIRD Probe qPCR Mix kit (Toyobo, Osaka, Japan) and PikoReal 96 system (Thermo Fisher Scientific, Waltham, MA, USA) using 40 ng of extracted genomic DNA according to the manufacturer’s protocols. To sequence SLC2A9 exons, 100 ng of genomic DNA were amplified in a 50-μL volume containing 1.25 U of PrimeSTAR GXL DNA Polymerase (TaKaRa Bio, Siga, Japan), 200 μM dNTPs, and the primer pair (0.4 μM). Amplified PCR products were purified using 1% agarose gel in 1 × TAE buffer and a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). DNA sequencing was performed using an Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).
In total, 2 exons of ATP-binding cassette subfamily G member 2 (ABCG2) and 13 exons of solute carrier family 2 member 9 (SLC2A9/GLUT9 variants 1 and 2) were analyzed. To analyze two common SNPs of ABCG2 (rs72552713 and rs2231142), we performed TaqMan-based probe qualitative real-time PCR for genotyping using a THUNDERBIRD Probe qPCR Mix kit (Toyobo, Osaka, Japan) and PikoReal 96 system (Thermo Fisher Scientific, Waltham, MA, USA) using 40 ng of extracted genomic DNA according to the manufacturer’s protocols. To sequence SLC2A9 exons, 100 ng of genomic DNA were amplified in a 50-μL volume containing 1.25 U of PrimeSTAR GXL DNA Polymerase (TaKaRa Bio, Siga, Japan), 200 μM dNTPs, and the primer pair (0.4 μM). Amplified PCR products were purified using 1% agarose gel in 1 × TAE buffer and a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). DNA sequencing was performed using an Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).