Fig. 4. NANOG is upregulated upon early differentiation. (A) The expression pattern of NANOG during differentiation was examined by real-time RT-PCR. Four stages of in vitro differentiation were examined: ES, EB2d, EB10d and EB30d. Shown is the abundance of NANOG transcript normalized to GAPDH levels per sample. Error bars represent s.e.m. (B) The expression pattern revealed by real-time RT-PCR was verified by semi-quantitative RT-PCR under non-saturating conditions. The temporal expression pattern of NANOG was compared with that of OCT4, which, unlike NANOG, does not show elevation of expression during differentiation. NTC (no templates control) was used as a negative control. GAPDH was used as positive control.