Multiplex Droplet Polymerase Chain Reaction to Detect DNA Methylation in Leukocytes

For droplet generation, mix 1 microliter of bisulfite-converted DNA with freshly-prepared probe master mix in a PCR tube, and collect the sample with a brief centrifugation. Use PEEK fittings to connect disposable fluidic tubing to two 250-microliter volume precision glass syringes, and pre-fill one precision glass syringe with 250 microliters of carrier oil containing 5% fluoro-surfactant, and one precision glass syringe with 50 microliters of carrier oil.

When both of the syringes have been loaded, load 100 microliters of the PCR mix into the syringe of carrier oil, and place a droplet microfluidic device onto the stage of an upright light microscope equipped with a high-speed camera.

For the observation and recording of droplet formation in real time, place the pre-filled syringes onto a programmable syringe pump, and use PEEK union with fittings to connect the tubing of the syringes to the tubing of the respective inlet channels of the droplet microfluidic device.

Place the tubing from the outlet of the droplet generator to the inside of a 0.5-milliliter PCR tube, and adjust the syringe pump flow rate to 2 microliters per minute. Do allow the droplet size to stabilize before collecting the resulting emulsion. Slowly and carefully collect the emulsion from the top of the tube, and transfer 75 microliters of the solution to a 200-microliter PCR tube for thermal cycling.

Then, confirm that the oil content in the PCR tube closely matches the volume of the dispersed phase to prevent coalescence of the droplets during thermal cycling, and place the 200-microliter tube into the thermal cycler.