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Maiocchi et al. Vessel Plus 2023;7:27 https://dx.doi.org/10.20517/2574-1209.2023.69 Page 5 of 19

Following PCR, remove the plate from the thermocycler and let it rest at room temperature for 5 min. Do
not freeze. Perform droplet reading within 24 h following the instructions for the Bio-Rad QX200 Droplet
Reader using the official Bio-Rad QX200 Droplet Reader Instruction Manual.

After completing the steps in the instruction manual, record the actual copy number of every lot. When
selecting a spike-in dilution, it is important to choose a robust dilution that does not oversaturate or fall
outside of the linear dynamic range of the targets of interest. For the miRNA targets presented in this
technical note, we recommend choosing a spike-in dilution that is approximately 500-1,000 copies per µL.

Figure 1B is a representative one-dimensional analysis of 0.1-0.0001 ng/µL miR-39 dilutions and the
respective NTC. The green dots represent VIC positive (miR-39) particles, while the grey dots represent
droplets with no fluorescence. To demonstrate that all analyses are performed within the linear dynamic
range of fluorescence detection, Figure 1C represents the ratio of miR-39 positive to negative particles.
Measured concentrations of miR-39, in copy per µL, are reported in triplicate in Figure 1D. The average of
these triplicates is used to record copy numbers for each lot of miR-39 cDNA spike-in prepared.

From the dilution selected, prepare small-volume aliquots. Vortex the large volumes for 30 s, followed by a
30-s rest on ice. Repeat this process 3 times, then aliquot into microcentrifuge tubes, and store at -80 °C
until further use. Note: once an aliquot has undergone 3 freeze-thaw cycles, it should no longer be used. When
ready, thaw the aliquots on ice or at 4 °C.

2. Blood collection, plasma isolation, and long-term storage [Figure 2A]:

To collect and isolate plasma from human blood, perform the following steps. First, a trained health care
professional collects 5 mL of peripheral venous blood using an 18-gauge angiocatheter into a prelabeled
13 mm × 75 mm, plastic, BD Vacutainer® EDTA-coated tube (K2 EDTA 7.2 mg, catalog number: 367861).
Note: EDTA is the recommended anticoagulant for miRNA quantification using this protocol. Immediately
after collection, invert the tube several times until it is thoroughly mixed and store it on ice for no more
than 2 h. If not stored on ice, centrifuge the collected blood sample immediately at 2,500× g for 15 min at
room temperature. This process separates the plasma from the rest of the whole blood components.
Carefully collect the topmost layer (the plasma) of the supernatant and transfer it to a sterile, nuclease-free
15 mL conical tube. Note: It is crucial to avoid disrupting the middle layer, known as the buffy coat. To do so,
leave approximately 1 cm of plasma above it.

Centrifuge the transferred plasma at 10,000× g for 15 min at room temperature, further purifying it and
removing any unwanted cellular debris. Transfer only the topmost soluble volume into a new 15 mL conical
tube. Note: Leave the bottom ~100 μL of plasma in the tube to avoid any remaining impurities. Next, aliquot
desired volumes into appropriately labeled, freezer-safe, nuclease-free microcentrifuge tubes. Note: a
minimum aliquot volume of 250 μL is recommended for this protocol. Snap freeze plasma fractionations in
liquid nitrogen or a slurry of dry ice and isopropanol and transfer to -80 °C for long-term cryo-preservation.
This ensures the integrity of miRNAs in the collected plasma samples for later use.

While performing the above steps, it is important to monitor for hemolysis. Hemolysis is the rupture of red
blood cells and the release of hemoglobin. Hemolyzed samples will appear pink or red in color and must be
excluded from analysis.