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Fig. 3 | BMC Biology

Fig. 3

From: Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium

Fig. 3

Profiling the differential expression of RNA between hPSCs and the differentiated endothelial cells using STA. a Fluorescence-activated cell sorting for CD31+/CD34+ cells. hESCs (20,000) were induced for 2 days into mesoderm. The induced mesodermal cells (20,000) were replated into a vascular mixture for another 3 days before sorting. The cells in the boxed area were seeded directly into 2 μl of lysis buffer for STA. b Unsupervised principal component analysis (PCA) based on the expression of total RNA of 100 hESCs and the differentiated endothelial cells. Genes (GENCODE v22) with summed counts >20 across six samples were rlog-transformed with DESeq2 (blind = TRUE), and the output was subject to PCA with default parameters. c Unsupervised heat map of the data in b. The top 150 variable genes of the rlog-transformed counts in b served as the input for heatmap3 analysis with default parameters. Blue, red, and green clusters indicate genes enriched in hESCs, endothelial cells, and the TW1 cell line, respectively. d Volcano plot showing the differential expression of miRNA between 100 hPSCs and endothelial cells. miRNA (GENCODE v22) with summed counts >1 across six samples were included for differential-expression analysis with DESeq2. Colored dots indicate log2 fold change >1 and an adjusted p value <0.05. miRNAs for further validation and in miR-302/367 cluster are highlighted with a black border. qPCR (e) and representative denaturing PAGE (12%) of the amplified products (f) of miR-302c, miR-367, miR-498, miR-515, miR-519e, miR-19b, miR-92a, miR-24-2, miR-126-3p, miR-126-5p, and miR-887. DF19-9-7T cells and their day-2 differentiated mesodermal (MES) and day-5 endothelial (END) progenies were lysed for STA (15 cycles) and qPCR (e) or semi-quantitative PCR (f). The differentiation procedure was performed as in a, and 100 cells at the mesoderm and endothelial stages were harvested directly for STA (15-cycle preamplification). One-hundredth of the preamplified and purified eluents was used for qPCR and PCR. The optimal cycle numbers (threshold cycle plus 4) for semi-quantitative PCR in f were based on the qPCR analysis in e. One hundred undifferentiated DF19-9-7T cells served as the reference sample. PSMD4 served as a loading control for analyses with qpcR. g Scatter plots correlating the expressions of the RNA-Seq data with the qPCR quantifications. y-axis represents the regressed means of the hESC data by DESeq2 in d. x-axis (μ) represents the relative expression of the respective miRNAs normalized by the PSMD4 of the DF19-9-7T cell line in e. Eff: efficiency; cpD2: threshold cycle; left panel: hPSC; right panel: differentiated endothelial cells

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