Multiplex Droplet Polymerase Chain Reaction to Detect DNA Methylation in Leukocytes

Overview

This video demonstrates the multiplex droplet polymerase chain reaction technique for the simultaneous quantification of methylated DNA regions in different white blood cell populations as an early biomarker for clinical diagnosis and prognosis.

Protocol

1. Droplet generation and PCR

NOTE: Table 1 outlines information on the forward and reverse primers along with the double-quenched hydrolysis probes for C-LESS, CD3Z, and Foxp3 genes, which are required for the multiplex amplification of demethylated gene targets.

1. Prepare the master mix as described in Table 2.
2. Thaw all components of the master mix except for the enzyme mix. Mix the master mix thoroughly by pipetting up-down, and spin down briefly.
3. Add the appropriate volume (1 μL) of bisulfite-converted DNA to the master mix in a PCR tube. Mix the reaction by pipetting up-down and spin down briefly.
4. Connect disposable fluidic tubing (I.D. 0.25 mm, O.D. 1.6 mm) to two precision glass syringes (250 μL volume) using PEEK fittings.
5. Prefill one precision glass syringe with 250 μL of carrier oil containing 5% fluoro-surfactant.
6. Prefill another precision glass syringe with 50 µL of carrier oil before loading 100 µL of the PCR mix to ensure dispensing of the entire sample volume during emulsification.
7. Set up a droplet microfluidic device on a stage of an upright light microscope equipped with a high-speed camera to observe and record droplet formation in real-time.
8. Place the prefilled syringes onto the programmable syringe pump and using PEEK union with fittings (see Table of Materials), connect the tubing of the syringes to the tubing of the respective inlet channels of the droplet microfluidic device.
9. Place the tubing from the outlet of the droplet generator inside a 0.5 mL PCR tube.
10. Adjust the flow rate of the syringe pump to 2 µL/min and allow for the droplet size to stabilize before collecting the resulting emulsion.
11. Collect the emulsion and transfer 75 μL to a 0.2 mL PCR tube for thermal cycling.
12. Ensure that the oil content in the PCR tube closely matches the volume of the dispersed phase in order to prevent the coalescence of the droplets during thermal cycling.
13. Place the 0.2 mL PCR tube in a thermal cycler and perform the cycling protocol as follows: preheating at 95 °C for 5 min, then 45 cycles of denaturation at 95 °C for 15 s, and annealing/extension at 60 °C for 30 s.

Table 1: Primer and hydrolysis probe design.

FOXP3 Forward	GGG TTT TGT TGT TAT AGT TTT TG
FOXP3 Reverse	TTC TCT TCC TCC ATA ATA TCA
CD3Z Forward	GGA TGG TTG TGG TGA AAA GTG
CD3Z Reverse	CAA AAA CTC CTT TTC TCC TAA CCA
C-LESS Forward	TTG TAT GTA TGT GAG TGT GGG AGA GA
C-LESS Reverse	TTT CTT CCA CCC CTT CTC TTC C
FOXP3 Probe	/56-FAM/CA ACA CAT C/ZEN/C AAC CAC CAT /3IABkFQ/
CDZ3 Probe	/56-HEX/CC AAC CAC C/ZEN/A CTA CCT CAA /3IABkFQ/
C-Less Probe	/56-CY5/CT CCC CCT C/ZEN/T AAC TCT AT/3IABkFQ/

Table 2: Master mix recipe for mdPCR.

Reagent	Stock Solution	Master Mix Volume	Working Concentration
Tris-HCl	1 M	2 µL	20 mM
KCl	1 M	10 µL	100 mM
MgCl2	25 mM	16 µL	4 mM
C-LESS Primers	10 µM	10 µL	1 µM each
CD3Z Primers	10 µM	10 µL	1 µM each
FOXP3 Primers	10 µM	10 µL	1 µM each
C-LESS probe	10 µM	5 µL	500 nM
CD3Z probe	10 µM	5 µL	500 nM
FOXP3 probe	10 µM	5 µL	500 nM
HotStarTaq DNA Polymerase	5 Units/µL	8 µL	0.4 Units/µL
Bisulfite-treated DNA Target	Variable	Variable	Variable
Nuclease-Free Water		Variable
Total Volume		100 µL

Materials

Name	Company	Catalog Number	Comments
PCR tube	Bio-Rad, Mississauga, ON	TFI0201
Fluoro-surfactant	RAN Biotechnologies, Beverly, MA	008-FluoroSurfactant
Disposable fluidic tubing	Dolomite	3200063
Disposable fluidic tubing	Dolomite	3200302
PEEK fittings	IDEX Health & Science, Oak Harbor, WA	P-881
Carrier Oil	3M, St Paul, MN
Upright light microscope	Nikon, Melville, NY