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Fig. 2 | BMC Research Notes

Fig. 2

From: Quantification of circulating microRNAs by droplet digital PCR for cancer detection

Fig. 2

ddPCR quality control parameters for an accurate quantification of circulating miR-320a. All the 10 serum samples (5 from health-donors and 5 from ovarian cancer patients) were submitted to a ddPCR reaction whereas the quality control parameters were evaluated. a Representative 1D plot showing the effect of the annealing temperature at 58 °C and 1 µL of LNA primer volume on the amplitude of positive (blue) and negative (gray) droplets. No template control (NTC) has not positive droplets. b All samples had more than 10,000 total droplets detected even in a single-plex (gray bars), and a minimum  number of positive droplets ≥ 3 was considered to call a positive sample for miR-320a detection (pink bars). NTC (Negative Template Control) had 0 droplets indicating no false-positive droplets calling. c, d Bi-dimensional plots showing the “raining droplets” (blue droplets between 3000–8000 amplitude), a typical appearance of positive droplets detected for miRCURY LNA primers combined with EvaGreen-based assays. Around 10,000 amplitude is possible to observe one accumulation of miR-320a positive droplets in ovarian cancer case (d) (OV16) compared to the control (c) (C1). e Absolute quantification (concentration in copies/µL) of miR-320a for all 10 samples. f Mann–Whitney test was applied for all 10 samples for miR-320a quantification after normalization considering the geometric mean of cel-miR-39–39 and UniSp6. The miR-320a expression profile was able to distinguish health-controls from ovarian cancer serum samples (P = 0.037). C Health-controls, OV ovarian cancer patients, RQ Relative quantification. Error bars = Poisson 95% confidence interval. Representative data from 2 independent experiments

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