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Page 6 of 19 Maiocchi et al. Vessel Plus 2023;7:27 https://dx.doi.org/10.20517/2574-1209.2023.69

Figure 2. Schematic diagram of the microRNA quantification workflow. (A) Blood collection, plasma isolation, and long-term storage.
Peripheral venous blood is collected in BD Vacutainer® EDTA-coated tube and plasma is separated by centrifugation (2,500× g, 15 min,
room temperature). Plasma supernatant is removed and centrifuged again and supernatant is aliquoted for storage at -80 °C. (B) Small
RNA Isolation from Plasma. microRNA is isolated according to the manufacturer’s instructions of the Qiagen miRNeasy Serum/Plasma
Advanced Kit. (C) Small RNA quantification and concentration normalization. The concentration (ng/μL) of eluted microRNA is
quantified using the Qubit microRNA assay, according to manufacturer’s instructions. Concentration is subsequently adjusted to
122.55 ng/μL for all samples. (D) Spike-in addition and microRNA specific cDNA generation. miR-39 is exogenously spiked into the
microRNA sample, and target-specific cDNA generation is performed utilizing TaqMan microRNA Reverse Transcription Kit following
manufacturer’s instructions. miR-39-specific and target-specific cDNA generation are performed separately to avoid cross-reactivity.
(E) Droplet generation and microRNA specific PCR amplification. miR-39 cDNA and target-specific cDNA are mixed together with
ddPCR supermix (no DUTP), and respective FAM and VIC miR-39 and target-specific cDNA amplification primers. Samples are
transferred to Bio-Rad droplet generator microfluidic chips alongside droplet generator oil and transferred to the QX200 Droplet
Generator. Droplets are transferred to a deep-well PCR plate, followed by PCR amplification. (F) Droplet reading and data analysis. The
samples are read by the Bio-Rad QX200 Droplet Reader and analyzed with Bio-Rad QuantaSoft software. All appropriate instructions in
user manuals are adhered to.

Plasma may be stored at -80 °C for up to two years. Once a single aliquot of plasma is thawed, you must
perform all operations through the cDNA generation process. After the cDNA generation process has been
completed, you can preserve specimens long-term (for up to one year) in cryopreservation.

3. Small RNA isolation from plasma [Figure 2B]:

A stepwise protocol is available in the supplement [Supplementary File 2].

We utilized the Qiagen miRNeasy Serum/Plasma Advanced Kit (Catalogue # 217204) as it has been
reported to be a robust and reproducible method for miRNA purification from plasma . We confirmed
[9]
that in our hands, we found that we consistently isolated sufficient amounts of miRNA. We follow all
instructions from the manufacturer, which can be found in the user manual. The following is a summary
that includes precise numerical values for optimized reaction volumes and conditions.

MiRNA may be extracted from plasma by performing the following: First, retrieve the aliquoted 250 µL
plasma samples from the -80 °C freezer and thaw them either on ice or at 4 °C. Thawing takes
approximately 1 h. Vortex, then transfer 240 µL of plasma into a 2 mL microcentrifuge tube and centrifuge
at 1,000× g for 10 min at 4 °C. Then, pipette 200 µL of the plasma supernatant to a new 2 mL
microcentrifuge tube and add 60 µL of buffer RPL, vortex for 5 s, and then incubate for 3 min at room
temperature. Following incubation, add 20 µL of buffer RPP to each tube, vortex for 30 s, and incubate again
for 3 min at room temperature.