To quantitatively analyze multiple marker gene expressions at multiple time points during differentiation (days 0, 3, 7, 14 and 21 of differentiation), we performed real-time PCR analysis. Total RNA was prepared using TRIzol reagent and PureLink RNA mini kit. cDNA from total RNA was generated using SuperScript II reverse transcriptase and Oligo d(T) primers. For quantitative analysis of the expression level of mRNAs, real-time PCR analyses were performed using the DNA Engine Opticon system and SYBR Green I assays. Primers were designed using the MacVector software (primer sequences are available upon request). PCR was carried out in a 25 µl volume containing 0.5 mM of each primer, 0.5 × SYBR Green I, and 1 µl of cDNA and performed with conditions of a denaturation of 95 °C for 2 min, followed by 50 cycles of amplification (95 °C for 30 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
