Gene Expression Analysis of hESC Differentiation Figure 3. Confirmation of array data with quantitative real time RT-PCR for transcripts that were changed during differentiation. Real time PCR data (A) for stem cells specific transcripts (POU5F1, SOX2, ALPL and NANOG) that were down regulated during neural differentiation compared with array data (B). The expression of neural progenitor specific transcripts (FOXA2, Nestin and PAX6) and maturation markers (TH, HOXB5, HOXA5 and HOXA2) also compared. In all of the above transcripts, real time RT-PCR data confirmed the array results, just in the case of ACTB gene, that has a background expression in differentiated neurons in the array data, the real time data revealed that ACTB was down regulated in the neural differentiation. SOX1 expression also was not significantly changed in array (data was not shown) but has a background expression in differentiated neurons in the real time PCR data. Expression data were analyzed with the DDCT method. doi:10.1371/journal.pone.0022856.g003 Figure 4. The diagram represents up- and down-regulated genes at different stages compared with hESCs. Significantly expressed transcripts (P,0.05, FC$1.5) used as an input transcripts for clustering. The number of differentially expressed genes increased as differentiation progressed. doi:10.1371/journal.pone.0022856.g004 PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22856