s, 55 °C for 30 s, 72 °C for 30 s, and 79 °C for 5 s). Primer dimers were melted at 79 °C before the fluorescent signals after each cycle were measured. The mRNA expression level of each gene was normalized using the housekeeping gene GAPDH as an internal control. The relative values were calculated by setting the normalized value of control as 1. Real-time PCR analysis under these optimal conditions showed that expression of pluripotent stem cell markers Nanog and Oct4 was down-regulated by day 3, and expression of ventral telencephalic markers Nkx2.1, GSX2, DLX2 became evident starting on day 14, followed by expression of postmitotic MGE marker Lhx6 on day 21 (Fig. 5).
