TaqMan qRT-PCR on genes of interest shows preferentially silenced or decreased expression in MLL-r primary samples in comparison with MLL-wt ALL primary samples and in normal controls in comparison with published gene expression microarrays validating results. The 12 genes described in Tables 2 and 3 were analyzed by qRT-PCR on the HELP primary samples and 2 peripheral blood samples from healthy donors (PB). The results are summarized in the heat map (A), which has the genes of interest represented by rows and individual samples, by columns. Each box shows the ΔC_t (gene C_t − GAPDH C_t), such that higher numbers indicate lower RNA expression. In addition, the heat map is color-coded based on its biology as described in the key. The color code was determined by natural peaks in a histogram of all C_ts in the cohort. This histogram is also shown in the key. The MLL-r samples show a greater number of silenced genes and more underexpressed genes than do the other leukemias or normals. C_t values were then converted to relative gene expression using the 2^(−ΔCt) method, and MLL-r expression levels were compared with MLL-wt and normal control levels (raw data in supplemental Table 1). By dot plot, many of the genes show statistically significant differences in gene expression between groups. In analysis, FLT3 (B) was statistically significantly up-regulated in the combination of MLL-r infant leukemia (M) and hyperdiploid (H) leukemia compared with normals; in addition, MEIS1 (C) was shown to be up-regulated in MLL-r leukemia; both of these phenomena are consistent with published literature. Finally, there was statistically significant down-regulation on FHIT (D) and DAPK1 (E) in MLL-r leukemia compared with “other leukemias” and normal controls. (F) To further validate our qRT-PCR results, we compared our data to 3 published gene expression microarrays. Based on our qRT-PCR results (A-E and supplemental Table 1), we predicted that the genes FLT3, MEIS1, and HOXa9 were up-regulated in MLL-r ALL in comparison with MLL-wt ALL, that the genes DAXX, LIFR, and CASPASE9 had equivalent expression in MLL-r ALL and MLL-wt ALL, and that the genes FHIT, DAPK1, CCR6, and HRK were down-regulated in MLL-r ALL in comparison with MLL-wt ALL. (G) Based on 3 independent gene expression microarrays using 2 different Affymetrix platforms (HG-U95A and HG-U133A) published by Armstrong et al (A),¹¹  Yeoh et al (Y),¹⁴  and Ross et al (R),¹³  we confirmed the predictions.