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Chapter 5104Supplemental Figure S2: Thermal gradient for primer assay optimization. The dd qPCR is performed at 8 different annealing temperatures: 62.0%u00b0C, 61.2%u00b0C, 60.0%u00b0C, 58.1%u00b0C, 55.8%u00b0C, 53.9%u00b0C, 52.7%u00b0C, 52.0%u00b0C. The following primers are used: miR-200c-3p, miR-375, miR-21-5p, miR-148a-3p, miR-141-3p, miR-146a-5p and miR-218-5p. Droplet are spaced out individually for fluorescence reading by the droplet reader. Positive droplets, which contain at least one copy of the measured miRNA exhibit increased fluorescence compared to negative droplets. Fluorescence in two channels is then measured for individual droplets. Each droplet from a sample plotted on the graph of fluorescence intensity vs. droplet number. The droplets in blue represent the positive droplets and the droplets in grey represent the negative ones. miR-148a-3p at 58.1%u00b0C is not visualized due an insufficient number of formed droplets (<10,000).