Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics

This protocol presents a flexible multiplex droplet PCR workflow that allows precise differential leukocyte counting based on epigenetic methylation markers. This flexibility may facilitate the translation of mdPCR towards clinical use. This method provides results that closely query to those obtained using immunofluorescent staining methods.

Unlike immunofluorescent staining, however, this method doesn't require fresh blood samples or costly antibodies. In hematological diagnostics, differential leukocyte can serve as indicators for a spectrum of diseases, including infection, inflammation, anemia and leukemia, and is being considered as an early prognostic cancer biomarker. Demonstrating the procedure with Abdelrahman Elmanzalawy will be Christina Nassif, a technical officer from our team at NRC's Medical Device Research Centre.

Begin by thoroughly mixing 20 microliters of protein kinase K and 400 microliters of lysis binding buffer containing magnetic beads with freshly thawed human peripheral blood mononuclear cells in 100 microliters of PBS. After a five-minute incubation at room temperature, place the tube on a magnetic rack for one to two minutes before removing the supernatant. With the tube removed from the magnet, re-suspend the DNA bead complex in 600 microliters of wash buffer one to remove any non-specifically bound beads.

Place the tube back onto the magnet to allow removal of the supernatant, before washing the cells in 600 microliters of wash buffer two as just demonstrated. After removing the supernatant, allow the tube to air-dry on the magnet for one minute before adding 100 microliters of elution buffer to the tube with thorough mixing. Then, place the tube on the magnetic rack for one to two minutes to separate the magnetic beads from the alluded DNA, and transfer the purified DNA solution to a new tube.

For bisulfite conversion of the purified DNA, transfer 20 microliters of the alluded DNA sample to a PCR tube, and add 130 microliters of conversion reagent to the tube. After thorough mixing, briefly spin down the tube contents and amplify the DNA on a thermal cycler. At the end of the cycle, add 600 microliters of binding buffer to an ion chromatography column placed in a collection tube, and add the DNA to the column.

Invert the tube several times to mix, and centrifuge for 30 seconds at full speed. Discard the collected flow through, and add 100 microliters of wash buffer to the column. Centrifuge the column and discard the flow-through again as demonstrated.

Add 200 microliters of Desulfonation Buffer to the column for a 15 to 20 minute incubation at room temperature, before centrifuging the sample and discarding the flow-through as demonstrated. Next, wash the column two times with 200 microliters of wash buffer per wash. After the second wash, transfer the column to a new 1.5 milliliter collection tube, and add 100 microliters of PCR grade water to the membrane of the column.

Then, elute the DNA by centrifugation of the column for one minute at full speed. For droplet generation, mix one microliter of bisulfite-converted DNA with freshly prepared probe master mix in a PCR tube, and collect the sample with a brief centrifugation. Use peak 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%flouro 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 prefilled syringes onto a programmable syringe pump, and use peak 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 two microliters per minute, to 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.

For fluorescence imaging of the emulsified sample, transfer the PCR emulsion into a 50-micrometer deep borosilicate capillary tube with a rectangular profile, to allow arrangement of the droplets into a close packed monolayer for imaging. After fixing and sealing the capillaries onto a microscope slide, load the slide onto the stage of the inverted microscope. And in the microscope imaging software, select acquire and live fast, to start real-time camera acquisition.

Then, observe the sample under bright field and fluorescence microscopy. Following droplet generation and thermal cycling, the droplets can be introduced into a glass capillary with a one-millimeter width and a 50-micrometer height. To obtain a monolayer distribution of the droplets that is ideal for fluorescence image acquisition, images can then be recorded for each wavelength.

After image analysis, the fluorescence intensity of all of the droplets in their respective fluorophores can be plotted to establish the threshold of the positive and negative droplets. After identification and counting, the copies per droplet values can be calculated, and the percentage of CD3+T-cells, and CD4+25+regulatory T-cells can be determined based on the methylated CD3Z and FOXP3 copies respectively, with respect to the copies per droplet of the total cells or C-less gene. The percent values can then be compared to those obtained through immunofluorescence imaging using the appropriate antibodies.

I think the emulsion stability from the droplet generation through the thermal cycling and final droplet imaging steps is crucial for obtaining precise, reliable and reproducible quantification results. MD PCR customization through tailored PCR mix formulation can be used for a number of applications including cancer research, infectious disease diagnosis and analytics at the single cell level.