Global changes in microRNA expression during megakaryopoiesis. (A) “Volcano plot” showing differential expression of microRNAs in Ter119^– fetal liver hematopoietic progenitors versus megakaryocytes, prepared as described in Figure 1. The x-axis indicates the fold change in megakaryocytes relative to progenitors, and the y-axis is the P value calculated by the paired significance analysis of microarrays method, an alternative to the Student t test.³⁵  Each dot represents 1 microRNA. MiRNAs that were investigated further in validation studies (panels B-E) are indicated by name. MicroRNAs with at least 2-fold change and P value less than .05 are shown as solid black circles and are described further in Table 1. (B) Real-time Q-PCR analyzing expression of selected miRNAs indicated in panel A that are repressed during megakaryopoiesis. MiRNA expression was compared in Ter 119^– fetal liver hematopoietic progenitors (assigned a relative value of 1), megakaryocytes generated after 3 days of culture (Meg), and Ter119⁺ erythroblasts (Ery). MiRNA expression was normalized to U6 snRNA. (C) Q-PCR analysis of selected miRNAs found to be induced during megakaryopoiesis, performed as outlined for panel B. (D) Q-PCR analysis of miR-146a in lineage-depleted hematopoietic progenitors (lin^–; assigned a relative value of 1), cultured megakaryocytes (Meg), and Ter119⁺ erythroblasts. The last 2 bars show miR-146a expression in human CD34+ progenitors (assigned a relative value of 1) and their megakaryocyte (Meg) progeny after 14 days of culture in the presence of thrombopoietin. MiRNA expression in all samples was normalized to U6 snRNA levels. (E) Q-PCR illustrating the kinetics of miR-146a induction in fetal liver megakaryocyte cultures.