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Figure 4. Representative 2-dimensional ddPCR plots of cardiovascular disease related microRNAs. ddPCR plots are separated into 4
quadrants representing FAM(-), VIC(-) events (lower left), FAM(-), VIC(+) events (lower right), FAM(+), VIC(-) events (upper left),
and FAM(+) VIC(+) events (upper right). The analysis allows for quantification of total copy numbers, as well as a ratio of the
microRNA of interest specific events [FAM(+), VIC(+)] normalized to the spike-in of miR39, represented by FAM(-), VIC(+) events.

Table 1 details the expected values of all miRNA targets in healthy individuals with no significant medical
history. While no statistical differences were detected between healthy males and females,
Supplementary Table 1 has all levels demarcated by sex. These tables are provided to assist in the
independent corroboration of results using this method.

DISCUSSION
Accurate quantification of nucleic acids, particularly miRNAs, from blood plays a pivotal role in
understanding disease mechanisms, identifying biomarkers, and developing diagnostic approaches.
However, ensuring the reliability and reproducibility of quantitative results necessitates standardized
protocols for blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription,
droplet generation, PCR amplification, fluorescence reading, and data analysis. By standardizing these steps
and implementing appropriate quality control measures, researchers and clinicians can enhance the
robustness and validity of circulating miRNA quantification. To assist independent corroboration of
conclusions, this protocol provides comprehensive disclosure of all relevant experimental details required to