PCR for CACNA2D2 was conducted with TaKaRa LA Taq polymerase (TaKaRa, Shiga, Japan) by touch-down PCR: 94°C for 3 min, then 10 cycles of 94°C for 20 s, 60°C down to 50°C (−1°C/cycle) for 30s, 72°C for 1 min, then 25 cycles of 94°C for 20 s, 50°C for 30 s, 72°C for 1 min, then 72°C for 10 min and hold at 4°C. Primers used were forward 5’-AAGACGGATGGCCTCGTTA-3’ and reverse 5’-ACATATGGATGGCCAGTTGAA-3’. The second PCR was conducted using the HotGoldStar DNA polymerase (Eurogentec, Seraing, Belgium) at 94°C for 3 min, then 40 cycles of 94°C for 30 s, 65°C for 30 s, 72°C for 1 min and ending with 72°C for 1 min and hold at 4°C, using primers forward 5’-TCCAACATCACTCGGGCCAACT-3’ and reverse 5’-TTGTTGGCACAGGCCATCCACT-3’.
