Peripheral blood cells miRNA profiles.

<p>(A) Pie graph representation of the top 10 most abundant miRNAs in each cell type. (B) Unsupervised hierarchical clustering of miRNA expression profiles. The dendrogram was generated using the union of the 20 most abundant miRNAs (39 in total) in each cell type. Data from 24 samples was used in these analyses (one granulocyte sample was not analyzed due to technical issues). (C) The left heatmap is derived from the average of the miRNAs shown in panel B. For ease of comparison, the right set of heatmaps display primary miRNA profiles adjacent to corresponding profiles from transformed cell lines, Jurkat (T-lymphoblastic), Raji (B-lymphoblastic), U937 (monocytic), Meg-01 (megakaryoblastic) and K562 (erythroleukemic) cell lines. Each column in the heatmaps indicates the average log-ratio intensity data. * indicates probes with similar and indistinguishable sequence with the nCounter platform.</p

miRNAs DE by cell type.

<p>(A) The 10 most stable miRNAs across all cell types are shown for both NormFinder (left) and Coefficient of Variation (CV) methods (right). (B) Validation of NanoString-derived data for <i>miR-301a-3p</i>by qRT-PCR data. The X-axis represents the expression level of each miRNA normalized to <i>miR-30c-3p</i> using 2^−ΔCt method. Each point represents the mean ± SEM of 5 subjects for each cell type. (C,D) Validation of microRNAs DE by cell type. The NanoString-derived data for the indicated miRNAs was validated by qRT-PCR using RNA isolated from the 5 hematopoietic cell types from two different preparations of cells and RNA. qRT-PCR data was normalized and presented as in Figure 4B.P, platelets; T, T-cells; B, B-cells; G, granulocytes; E, erythrocytes.</p

Exploiting endogenous miRNAs to modify exogenous gene expression.

<p>(A) Illustration that <i>miR-125a-5p</i> was selectively reduced in the lymphocytic cell lines, Jurkat and Raji. (B) Schematic of reporter constructs used to assess effect of endogenous levels of <i>miR-125a-5p</i>. Constructs were engineered to contain 1, 2 or 4 <i>miR-125a-5p</i> binding sites or scrambled sequence controls. (C) Meg-01 cells were transfected with the indicated constructs. Luciferase repression was enhanced with more <i>miR-125a-5p</i> binding sites. (D) Meg-01, Raji, Jurkat and K562 cells were transfected with the 4xSC and 4x125 constructs. Luciferase was quantified and normalized to GFP for transfection efficiency. Data plotted as fold-expression compared to constructs with scrambled sequence. (E) Meg-01 cells were co-transfected with Luc-4x125 construct and control locked nucleic acid (LNA) or LNA that specifically inhibits <i>miR-125a-5p</i>. Jurkat cells were co-transfected with Luc-4x125 construct and control pre-miRNA or pre-<i>miR-125a-5p.</i> Data in panels C-E are mean ± SD of at least three independent experiments with two replicates each.</p

Peripheral blood cells miRNA profiles.

<p>(A) Pie graph representation of the top 10 most abundant miRNAs in each cell type. (B) Unsupervised hierarchical clustering of miRNA expression profiles. The dendrogram was generated using the union of the 20 most abundant miRNAs (39 in total) in each cell type. Data from 24 samples was used in these analyses (one granulocyte sample was not analyzed due to technical issues). (C) The left heatmap is derived from the average of the miRNAs shown in panel B. For ease of comparison, the right set of heatmaps display primary miRNA profiles adjacent to corresponding profiles from transformed cell lines, Jurkat (T-lymphoblastic), Raji (B-lymphoblastic), U937 (monocytic), Meg-01 (megakaryoblastic) and K562 (erythroleukemic) cell lines. Each column in the heatmaps indicates the average log-ratio intensity data. * indicates probes with similar and indistinguishable sequence with the nCounter platform.</p

Quantification of total RNA and miRNA in platelets, T-Cells, B-Cells, granulocytes and erythrocytes.

<p>(A) Average yield of total RNA from each cell type. (B) Average of miRNA fraction in the total RNA from each cell type. (C) Average miRNA content of each cell type. In (A–C) the box represents the 25th to 75th percentiles, the line in the box is the median and the whiskers represent minimum and maximum values. (E) Summary of comparisons across cell types in panels A–C (one tail t-test). P, platelets; T, T-cells; B, B-cells; G, granulocytes; E, erythrocytes. N = 5 for each of the 5 cell types.</p

Contribution of hematopoietic cell type to total miRNA content per volume of blood.

<p>Contribution of hematopoietic cell type to total miRNA content per volume of blood.</p

miRNA repertoire per blood volume unit.

<p>Note: each row totals 100%</p><p>* Probes with similar and indistinguishable sequence in this assay.</p

Additional file 2: Table S2. of N-BLR, a primate-specific non-coding transcript leads to colorectal cancer invasion and migration

Primer sequences used for qRT-PCR, RACE, siRNAs, and vector construction in this study. (XLSX 11 kb