Duplex Digital PCR for Simultaneous Quantification of Dual Genetic Markers

To begin this procedure, first, prepare 100 micromole per liter stock concentrations for all primers in molecular-grade water and probes in TE pH 8 buffer, as described in the protocol text. Next, prepare a master mix by mixing appropriate amounts of digital PCR mix, forward and reverse primers, the fluorescent probe, and nuclease-free water. Pipette up and down at least 10 times to mix, while taking caution not to introduce air bubbles in the solution.

Since dPCR master mix is more viscous than conventional qPCR master mix, it's necessary to use the mixing by pipetting technique in order to create a homogeneous solution. This allows for accurate dPCR quantification downstream.

To make the assay mixture for droplet generation for running samples in duplicate, pipette 36 microliters of the master mix into a regular PCR plate. To each of the 36-microliter master mixes, mix in 12 microliters of DNA template, leaving the corresponding replicate wells empty on the plate. Include positive controls to ensure the assay is running properly. Include No Template Controls or NTCs to ensure there is no contamination within the plate, and to set the fluorescent baseline later for data analysis.

Prior to setting up the droplet generator, mix the assay mixtures by using a multichannel pipette to pipette the mixtures up and down approximately 15 times. Ensure that the pipette tip stays within the liquid to avoid making excess bubbles within the mixture.

Next, insert cartridge one, containing eight wells, into a white cartridge holder and click the cartridge holder shut. Cartridge one is now firmly in place, and cannot be dislodged from the holder while generating droplets.

Using a multichannel pipette, gently transfer 20 microliters of the assay mixture into the middle position of the cartridge marked 'Sample' without introducing air bubbles. Pipette in 70 microliters of droplet generation oil to the left side of the cartridge marked 'Oil.'

Cover the cartridge with a gasket, making sure the gasket is flat and held evenly by the four ticks toward the edge of the cartridge. Press the green-lit button on the droplet generator to open the door and place the cartridge. Press the button again to close the generator. Once the door closes, the green button is dimmed and the door cannot be reopened.

Droplet generation will begin immediately and continue for approximately 1 minute.

While the droplet generation is in progress, place cartridge two in a second white cartridge holder, and prepare it in the same manner as for cartridge one. When the droplet generation is complete, the dimly-lit button will turn green.

Open the droplet generator door. Remove the white cartridge holder containing cartridge one and set it aside. Place cartridge two into the droplet generator.

Remove the gasket from cartridge one and discard. Do not unclick the white cartridge holder, as the action may break the newly-generated droplets.

Using a multichannel pipette set to 40 microliters, insert the tips into the third column of the cartridge marked droplets at a 45-degree angle, and slowly pipette up all the droplets. Transfer them to the final PCR plate by putting the pipette tip against the well wall approximately halfway down and expelling the droplet slowly.

In the same way, when the droplet generation for cartridge two is complete, remove the gasket from cartridge two, and transfer the generated droplets to the final PCR plate. Place a pierceable foil cover on top of the plate and place it on a plate sealer. Set the sealer to 180 degrees Celsius. Press "Play" on the sealer and seal for 10 seconds.

To begin this procedure, place the sealed final PCR plate in the thermal cycler. Use a thermal cycler that is compatible with the final PCR plate, and with a temperature ramping speed of 2 degrees Celsius per second.

Run the following thermal program: 10 minutes at 95 degrees Celsius followed by 40 cycles of 30 seconds at 94 degrees Celsius and 60 seconds at 60 degrees Celsius followed by a 10-minute hold at 98 degrees Celsius. Upon completion of cycling, transfer the plate to a droplet reader for automatic measurement of fluorescence in each droplet in each well.

Ensure the droplets are at room temperature before proceeding with droplet reading. Start by opening the accompanying software to set up the droplet reading. In the default "Setup" menu containing a schematic of an empty 96-well plate, double-click on well A1 to open the menu containing three sections -- "Sample," "Assay 1," and "Assay 2."

In the sample section, type the sample ID into the box labeled "Name" and check the box to the right marked "Apply." Next, click the dropdown menu labeled "Experiment." Choose "RED" for Rare Event Detection and click 'Enter.'

Move to the section denoted as "A1." In the "Name" section, fill out the assay and click 'Enter.' In the box below labeled "Type," click the dropdown menu, choose "Channel 1 Unknown," and click 'Enter.'

Move to the section denoted as "A2." In the "Name" section, fill out the assay and click 'Enter.' In the box below labeled "Type," click the dropdown menu, choose "Channel 2 Unknown," and click 'Enter.'

All the information from the previous steps is now present in well A1.

Name all subsequent wells containing droplets. To save total setup time, click 'Shift' or 'Control' to choose multiple wells simultaneously. When the digital depiction of the plate mirrors the physical plate, press "OK" at the bottom right of the menu. In the new menu that appears at the top of the plate schematic, under the "Template" section, choose "Save As" and name and save the plate.

To the left of the screen, click "Run" and select the appropriate Dye Set in the pop-out "Run Options" window. Data collection will initiate and is displayed in real-time time in the software.