Processing of RNA-sequencing data to establish genes with altered expression and significant pathways. (A) Venn diagram illustrating the overlap of DEGs identified across each ET subtype vs HV (control) (adjusted P ≤ .05). JAK2V617F vs HV (light blue), CALR vs HV (gray), and TN vs HV (red); all samples adjusted for subtype vs HV (purple). The χ² test reflects the significance of the overlap between DEGs of each comparison with the adjusted comparison. (B) Volcano plot of all genes, comparing all ET samples vs HV samples (adjusted). A total of 528 genes (gold) are significantly downregulated and 310 genes upregulated (purple). (C) Heatmap showing the clustering of samples based on their expression of the 838 DEGs. The gold bar highlights the downregulated genes and the purple bar highlights the upregulated genes in the ET samples. (D) Enrichment plot showing the activated (purple) and deactivated (gold) functional annotations as determined by IPA analysis. X-axis displays the activation Z score; the higher the score, the greater the activation, with a negative score indicating deactivation. Circle size is based on the –log10 P value for the enrichment of each annotated term. All terms have an enrichment P value <3.4e-03. (E) Ranked plot of the gene expression values for all genes involved in activated terms derived from IPA. The x-axis represents each gene. The y-axis is the mean log2(CPM +0.5) expression for each gene in the ET cohort (purple) and the HVs (gold). The plot centers around the ranked expression of each gene in the HV (from highest to lowest expression). The size of each red point represents the –log10 adjusted P value for how significantly differentially expressed the gene is in the ET cohort vs the HV cohort. The shaded background represents the gene expression quantiles (green = upper quantile [Q3 + Q4], yellow = mid/low quantile [Q2], red = lowest quantile [Q1]). (F) Boxplots illustrating the qPCR results, comparing the mean –delta cycle threshold (CT) vs reference gene for the HV samples (gold), all ET samples (purple), CALR samples (pink), JAK2 samples (blue), and TN samples (red). The purple bar plot illustrates the significance of the difference between the HV and the ET cohort as a whole as determined by a Student t test. The –log10 P value is used here; thus, the higher the bar, the more significant the difference to HV. *Probes used for these 3 genes may detect genomic DNA.