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Page 4 of 19 Maiocchi et al. Vessel Plus 2023;7:27 https://dx.doi.org/10.20517/2574-1209.2023.69

Reverse Transcription Kit from Applied Biosystems (Catalogue # 4366597). The following is a summary that
includes precise numerical values for optimized volumes. Begin by preparing a miR-39 cDNA master mix
using the following components per reaction: 0.15 µL of dNTP Mix, 0.19 µL of RNase Inhibitor, 1.5 µL of
10X RT Buffer, 1 µL of Multiscribe Reverse Transcriptase, 0.75 µL of miR-39 RT Primers (20×), and 10.41 µL
of water, making a total volume of 14 µL. Add 14 µL of the master mix to all tubes. Add 1 µL of each miR-39
template to their respective tubes. Next, prepare the Negative Template Control (NTC) cDNA reaction by
substituting the miR-39 template with 1 µL of water. Please note: make an additional 10% excess volume for
all reaction mixes to account for lost volume during routine pipetting.

Place all tubes in a thermocycler. Set the thermocycler to the following conditions: 5 min at 4 °C, 30 min at
16 °C, 30 min at 42 °C, 5 min at 85 °C, and hold at 4 °C forever; lid temperature of 105 °C. Store the cDNA
at -20 °C; ddPCR analysis must be performed within one year of cryostorage.

ddPCR analysis of miR-39 exogenous spike-in
Quantify the copy number of each miR-39 cDNA dilution. First, prepare a 20× concentration of miR-39
TaqMan probe (Applied Biosystems, catalog number: 4427975, Assay ID: 000200 VIC-MGB labeled). Note:
If necessary, dilute any probes supplied to a 20X concentration in water. To perform ddPCR analysis, follow
the instruction manuals for the TaqMan miRNA Assay from Applied Biosystems and the Droplet Digital
PCR Applications Guide from Bio-Rad. The following is a summary that includes precise numerical values
for optimized volumes. Begin by preparing a miR-39 ddPCR master mix using the following components
per reaction: 12.5 µL of ddPCR Supermix (no dUTP); 1.25 µL of miR-39 VIC-labeled Probe (20×); 6.25 µL of
water. The resulting total volume should be 20 µL. After vortexing the master mix, spin it down using a
tabletop centrifuge, and carefully pipette 20 µL into the corresponding reactions. Spin down the cDNA
using the tabletop centrifuge and add 5 µL of the appropriate cDNA to each reaction, resulting in a final
volume of 25 µL. Prepare a Negative Control (NC) ddPCR “blank” reaction by adding 5 µL of water in place
of the cDNA for a total volume of 25 µL.

Next, partition the reactions into droplets using the Bio-Rad Droplet generator. Place the DG8 droplet
generator cassette (Bio-Rad DG8 Cartridges Catalog number: 1864008) into the droplet generator case (Bio-
Rad Catalog number: 1863051), ensuring full closure and correct positioning of the plate. Using a multi-
channel pipette, gently mix and transfer 20 µL of the samples to the row labeled “sample” on the droplet
generator cassette. Subsequently, pipette 70 µL of droplet generator oil from a sterile trough into the row
labeled “oil” on the droplet generator cassette. Exercise caution to not introduce bubbles. Securely place the
rubber gasket on top of the droplet generator plate and generate droplets.

Once the droplet generator process is complete, the row marked as "droplets" on the plate should appear
cloudy. Carefully transfer 40 µL of the droplets by gently withdrawing the pipette tip from the bottom of the
well at a 35-degree angle, slowly withdrawing over a period of 5 s. Transfer the droplets into a deep, 96-well
plate (Bio-Rad Catalog number: 12001925) by gently touching the pipette tip to the side of the well at the
halfway point and dispense slowly, taking care not to introduce bubbles or rupture any droplets. Using a
PX1 PCR Plate Sealer (Bio-Rad), seal the 96-deep well plate with pierceable foil (Bio-Rad Catalog number:
1814040) following the manufacturer’s instructions (180 °C for 5 s). Verify air-tight seals over each well
opening by checking for complete circles impressed into the foil.

Perform PCR by placing the sealed plate into a deep-well 96-well thermocycler. Set the thermocycler
conditions: 95 °C for 10 min (ramp 2 °C/s); 94 °C for 30 s (ramp 2 °C/s); 60 °C for 1 min (ramp 2 °C/s);
repeat at step 2, 39 times; 98 °C for 10 min (ramp 2 °C/s); hold at 4 °C indefinitely; lid temperature: 105 °C.