miR expression during human thymocyte development. miR expression profiles were generated as described in “miR and mRNA expression profiling” from a dataset consisting of 6 samples each of normal thymus tissue, DP thymocytes, CD4⁺ SP thymocytes (SP_ CD4), and CD8⁺ SP thymocytes (SP_CD8). (A) Dendrogram based on miR expression profiles of thymic populations showing the hierarchical clustering of biologic samples. (B-C) Heatmap representations of feature expression, limiting to the subsets of miRs that were differentially expressed in the following comparisons: DP versus SP thymocytes (B) and SP CD4⁺ versus SP CD8⁺ thymocytes (C). Differentially expressed features were identified by Significance Analysis of Microarray analysis²³  with false discovery rate = 0.001; the heatmaps were generated with the Genesis software package (BRB array tools ver. 3.6 Genesis 1.7.2).²⁴  (D) Validation of miR expression profiles in thymocyte subpopulations by quantitative RT-PCR (P < .05; t test). Reported are results of TaqMan quantitative RT-PCR to detect miRs chosen among the top-regulated miRs during thymocyte transition from DP to SP stage. The level of expression of was normalized to that of the small noncoding RNA RNU44. Plotted are the average values ± SD of 3 independent experiments from 3 biologic samples for each population.