5-azacytidine increases γ-globin and decreases β-globin mRNA levels. (A) The in vitro differentiation system used in this research. (B) γ- and β-globin steady-state mRNA levels during in vitro differentiation. The first panel shows RNA levels on the same scale. The second panel shows mRNA levels normalized to the maximal expression for each mRNA to emphasize the different time courses of expression. (C) Effects of daily 300 nM 5-Aza on steady-state mRNA levels for the α-, β-, and γ-globin and the GATA-1 genes. CD34⁺ cells were from different normal donors were used for experiments in panels B and C. Each RT-PCR analysis was performed in triplicate. Error bars represent 1 SD. Microscopy was performed using an Olympus model CHT light microscope (Olympus Optical, Tokyo, Japan). Magnification of Figure 1A was 100× (10×/10×; numeric aperture of the 10× objective was 0.25). Images were acquired with a FujiFilm FinePix A345 digital camera (Fujifilm USA, Valhalla, NY). Images were processed using iPhoto version 6.0.6 (Apple Computer, Cupertino, CA).