Microarray present call analysis.

<p>A) Average number (Y-axis) and standard deviation of miRNA detected in blood samples of six healthy individuals under four different blood sampling conditions (X-axis). Blood was collected in EDTA blood collection tubes and subsequently transferred into PAXgene^TM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgene^TM blood RNA tubes. B) Venn diagram of miRNAs that were present in all six individuals under different blood sampling conditions. There were 201 miRNAs detected in all individuals under all conditions. No miRNA was detected both in blood that was directly collected in PAXgene^TM blood RNA tubes and in blood that was transferred into PAXgene^TM tubes 2 h after phlebotomy without also being detected in blood directly transferred or transferred 10 min after phlebotomy. C) Balloon plot of miRNAs that show a difference in frequency under the different blood sampling conditions. The X-axis shows the minimal number of individuals that are positive for a miRNA under one condition. The Y-axis shows the maximum number of individuals that are positive for a miRNA under one condition. The top left balloon denotes the 806 miRNAs that were not detected in any of the 24 samples (minimum and maximum of 0). The lower right balloon denotes the 201 miRNAs that were detected in all samples (minimum and maximum of 6). The orange shaded area presents 12 miRNAs that show a difference in frequency of at least three under one of the four conditions as compared to the other conditions. The largest difference was found for miR-769-3p indicated in the lower left corner that was positive in 6 samples under the 2h EDTA condition and not in any sample of the PAXgene condition.</p

Scatter plot of the mean coefficient of variation (CV) for the four blood sampling conditions versus the overall CV.

<p>Both miR-769-3p and miR-135a* showed a higher overall CV as compared to their average CV within each of the four conditions indicating a condition specific abundance of both miRNAs.</p

Box-plots of normalized abundance values of miR-769-3p for the 6 individuals under the four blood sampling conditions.

<p>The most obvious change of abundance of miR-769-3p was found in blood that was transferred in PAXgene^TM blood RNA tubes 2 h after phlebotomy.</p

FISH analysis of amplified loci.

<p>For each FISH analysis, a BAC or cosmid clone containing the indicated gene was Cy3-labelled (pink) and hybridized against fixed NHNP cells that were differentiated for either 2, 5 or 7 days. Amplifications are shown for <i>C1QL1</i> RP11-113A24 (5 days), <i>SOX13</i> RP11876H8 (5 days), <i>HDGF</i> RP11-66D17 (5 days), <i>CYP27B1</i> cosmid (2 days), <i>GINS2</i> RP11-118F19 (2 days), <i>CDK4</i> RP11-571M6 (2 days), <i>TP53</i> RP11-1081A10 (7days), <i>DIABLO</i> RP11-568C23 (7 days). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm. Notably, the degree of amplification various within each analysis due to the high heterogeneity of the amplifications in each cell population.</p

Detailed gene amplification analysis on human chromosome 16 and 12.

<p>Representative sections of log₂ ratio profiles for undifferentiated (0 d) NHNP cells and cells that were differentiated for 2 and 5 days. Base count is given on the x-axis and log₂ ratio on the y-axis for chromosome 16q24.1 (A) and 12q14.1 (C). Chromosomal localization of BAC probes used for FISH were indicated at the bottom of figures C and D. A <i>GINS2</i> specific BAC probe that was labeled in pink and a <i>CDH13</i> specific BAC probe that was labeled in green were hybridized simultaneously against fixed NHNP cells that were differentiated for 2 days. <i>GINS2</i> amplification is indicated as pink speckled fluorescence signals whereas the neighboring <i>CDH13</i> gene shows only single copy fluorescence signals (Bi). Neighboring cells with and without <i>GINS2</i> amplification are shown in Figure Bii. A <i>CDK4</i> specific BAC probe that was labeled in pink and a <i>XRCC6BP1/KUB3</i> specific BAC that was labeled in green were hybridized simultaneously against NHNP cells that were differentiated for 2 days. <i>CDK4</i> and <i>KUB3</i> amplifications were detectable as cluster of pink and green speckled fluorescence signals. <i>CDK4</i> specific signals spread over a more extended area than the <i>KUB3</i> specific signals (D). Nuclei were counterstained with DAPI (B). Size calibration bar = 5 µm.</p

Whole chromosome plots.

<p>A genome-wide view of the 10× window-averaged data at 25 kb resolution is displayed for NHNP cells at day 0, day 1, day 2 and day 5 during differentiation.</p

Overview on amplified chromosomal regions.

<p>Chromosome regions that overlap with gained chromosome regions of TCGA glioblastoma samples are indicated in bold. Likewise, genes that were used for FISH analysis are indicated in bold. Examples of glioblastoma-amplified genes were included for chromosomal regions amplified after 5 d of differentiation. Start and end point were according to NCBI36/HG18.</p

FISH and immunofluorescence analysis.

<p>FISH with <i>GINS2</i> specific BAC (pink) and simultaneous immunofluorescence staining with GFAP (green) revealed <i>GINS2</i> amplification in cells with beginning GFAP expression after 5 d of differentiation (A). FISH with <i>CDK4</i> specific BAC (pink) and immunofluorescence staining with GFAP revealed <i>CDK4</i> amplification in cells with beginning GFAP expression after 2 d of differentiation (B). FISH with <i>CDK4</i> specific BAC (pink) and immunofluorescence staining with Tubulin-ß-III-chain (green) revealed <i>CDK4</i> amplification in cells with beginning Tubulin-ß-III-chain expression after 11 d of differentiation (C and D). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm.</p

Correlation of gene content to chromosome region.

<p>The correlation is shown for human chromosome 16 with several amplified and one deleted region in NHNP cells after 5 d differentiation. The log₂ ratio profile of the 10× window averaged data is presented at 25 kb resolution (A). Fused lasso analysis is performed using the same data points (B). GC repeats, gene content and banding pattern is shown as indicated by Ensembl genome browser (C). Both the log₂ ratio profile and the fused lasso analysis indicate an overlap between amplifications and regions with high GC and gene content and an overlap between deletion and regions with low GC and gene content.</p

miRNAs with fold change >1.5 in the different comparisons.

<p>miRNAs in italics are not expressed in all 32 tested samples. Numbers in bold are statistically significant p-values.</p