Page 146 - Read Online

Maiocchi et al. Vessel Plus 2023;7:27 https://dx.doi.org/10.20517/2574-1209.2023.69 Page 9 of 19

For the no template control (NTC) reactions, combine 5.84 µL of the appropriate master mix with 9.16 µL
of water in place of cDNA. Please note: each miRNA-specific master mix requires an NTC to confirm
specificity during the data analysis phase.

Centrifuge the tubes at room temperature and remove any bubbles. If necessary, pop any remaining bubbles
with a clean pipette tip, ensuring all bubbles are eliminated. Transfer the tubes to a thermocycler set to the
following conditions: 4 °C for 5 min; 16 °C for 30 min; 42 °C for 30 min; 85 °C for 5 min; hold at 4 °C
forever; lid temperature 105 °C.

After thermocycling is complete, either proceed to the next step immediately or store cDNAs at -20 °C
within 24 h. Droplet generation and miRNA specific PCR amplification must be performed within one year
of cDNA generation.

6. Droplet generation and miRNA specific PCR amplification [Figure 2E]:

In this step, cDNAs will be combined with PCR reaction mixes, partitioned into approximately 20,000
nano-droplets, and then PCR amplified. First, ensure pre-formulated TaqMan miRNA Assays (Tm probes)
(Applied biosystems Catalogue # 4427975) are diluted to a 20X concentration in water. TaqMan miRNA
Tm probes are supplied in a single tube containing 1 probe and two primers. For this protocol: All miRNA
target specific probes are labeled with FAM-MGB while the miR-39 specific probe is labeled with VIC-MGB
for multiplexed detection.

Prepare the master mixes for each target by thoroughly mixing the ddPCR Supermix for probes (no dUTP)
(Bio-Rad Catalog number: 186-3024) by vortexing for 30 s. To make the master mix, add 12.5 μL of ddPCR
Supermix, then 1.25 μL of the VIC-labelled miR39 probe and 1.25 μL of the FAM-labelled target-specific
probe, to make a final volume of 15 μL of master mix per sample. Vortex the completed mixtures. Note: we
recommend preparing an additional 10% of the master mix to account for any liquid lost during routine
handling. Vortex each master mix and then centrifuge with a tabletop centrifuge; pipette 15 µL into the
appropriate reactions.

Centrifuge the cDNA at 500× g for 30 s. Transfer 5 µL of the appropriate cDNAs into each reaction (5 μL of
target-specific cDNA and 5 μL of cDNA of corresponding sample’s miR-39 reaction), resulting in a final
volume of 25 µL for each multiplex reaction. Make a negative control (NC) reaction by combining 10 µL of
water with 15 µL of the above master mix. Vortex for 30 s, then centrifuge and remove air bubbles with
clean pipette tips.

Next, in a sterile trough, pour enough droplet generator oil to generate droplets for all samples. Place the
droplet generator plate into the droplet generator case, ensuring that the case is fully closed and that the
plate is positioned correctly. Using a multi-channel pipette, gently mix and transfer 20 µL of the sample plus
the master mix solution to the droplet generator plate. Note: take care not to introduce bubbles; always
pipette the sample into the plate first, followed by the droplet generator oil. If there are empty wells in a row of
the DG8 cartridge, this results in failed droplet generation. If necessary, load 25 µL of the buffer control for
probes into any empty well of the DG8 cartridge.

Using a multi-channel pipette, transfer 70 µL of droplet generator oil (Bio-Rad Catalog number: 1863005)
from a sterile trough into the wells of the “oil” row on the droplet generator cassette, taking care not to
introduce bubbles. Place the rubber gasket over the top of the droplet generator plate, ensuring that it is