Multiplex Droplet Polymerase Chain Reaction to Detect DNA Methylation in Leukocytes

Abstract

Source: Malic, L., et al. Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics. J. Vis. Exp. (2020)

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 react....

Materials

List of materials used in this article

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