Molecular phenotypes of primitive erythroid and primitive/definitive multipotent colonies by real-time qRT-PCR. Day 9 hEB colonies were scored with primitive/definitive parameters as described in the text. Primitive erythroid (BFU-e-P, EryP) colonies, primitive mixed (MIXED-P) colonies, and definitive mixed (MIXED-D) colonies were pooled (2 to 3 colonies), and RNA was analyzed by qRT-PCR methods as described in text. “Fold change in expression from undifferentiated hEB cells” was calculated using the 2^–/ΔΔCT method as described in “Materials and methods,” based on the C_T (threshold curve) for each target gene and internal normalizations with actin. Expression of target genes was compared with levels in undifferentiated day 9 hEB cells prior to plating. (A) qRT-PCR analysis of key regulatory genes in primitive and definitive colonies. (B) qRT-PCR expression analysis of primitive (epsilon, zeta), fetal (alpha, gamma), and definitive adult (alpha, beta) hemoglobins. (C) qRT-PCR analysis of AML1 isoforms. (D) Corresponding agarose gels of PCR products obtained at linear phases of qPCR reactions. RNA from erythroleukemia cell line K562 and cord blood were used as positive controls for qRT-PCR reactions.