Figure 4.

(A) Eluted RNAs were converted to cDNA, and cDNA was amplified by TS-PCR. (B) Validation with quantitative RT–PCR of the DNA microarray data using each amplification method. Genes expressed at various levels in the non-amplification method, TS-PCR amplification method, and T7 in vitro transcription (IVT) amplification method were confirmed using quantitative RT–PCR. Quantitative RT–PCR was performed using the total RNA of these transcription factors. Gapd was used as the control. The same total RNA was used for quantitative RT–PCR and DNA microarray experiments. The ratio of the total target RNA levels was normalized by using those of the Tbp (Mus musculus TATA box binding protein) gene, and the fold change for each gene in the differentiated ES cells compared to the control ES cells was calculated. Error bars represent standard deviations of log (base 2) of ratio. (C) Amplified cDNA labeled with Cy3/Cy5 underwent self-self hybridization.