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Epigenetic markers are used for white blood cell (WBC) subtyping through the quantification of DNA methylation patterns. This protocol presents a multiplex droplet polymerase chain reaction (mdPCR) method using a thermoplastic elastomer (TPE)-based microfluidic device for droplet generation allowing for precise and multiplex methylation-specific target quantification of WBC differential counts.
A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBCÂ subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.
Analysis of white blood cells (WBCs) composition is among the most frequently requested laboratory tests in hematological diagnostics. Differential leukocyte count serves as an indicator for a spectrum of diseases including infection, inflammation, anemia, and leukemia, and is under investigation as an early prognostic biomarker for several other conditions as well. Gold standard in WBC subtyping involves immunostaining and/or flow cytometry both of which require costly, instability-prone fluorescent antibodies and are often highly dependent on operator proficiency in sample preparation. Moreover, this method is applicable to fresh blood samples only, such that the sa....
All the experiments performed in this study involving human samples were approved by the NRC’s Ethics Board and were done according to NRC’s policies governing human subjects that follow applicable research guidelines and are compliant with the laws in Québec, Canada.
1. Cell preparation
The TPE-based microfluidic droplet generator device was fabricated using the described protocol as shown in Figure 1. A transparency mask was used in photolithography to obtain silicon (Si) master. Soft lithography was performed to obtain an inverse PDMS replica of the Si master which was then used to fabricate the epoxy mold. Epoxy precursor was poured onto the PDMS and cured to crosslink and harden. This mold, representing the exact replica of the Si master was more resilient for subsequen.......
The presented experimental protocol and methods allow for in-house mdPCR using a fabricated TPE droplet generator, a thermal cycler, and fluorescence microscope. The fabricated device using soft TPE to TPE bonding affords hydrophobic surface properties that are uniform across all channel walls, such that the final device does not require any surface treatment for subsequent use as a droplet generator. This material has been routinely employed in point-of-care platforms that necessitate compatibility with high throughput .......
The authors acknowledge financial support from the National Research Council of Canada.
....Name | Company | Catalog Number | Comments |
Bio-Rad, Mississauga, ON | TFI0201 | PCR tube | |
RAN Biotechnologies, Beverly, MA | 008-FluoroSurfactant | Fluoro-surfactant | |
Silicon Quest International, Santa Clara, CA | |||
Oxford Instruments, Abingdon, UK | EMCCD camera | ||
Thermo Fisher Scientific, Waltham, MA | MA5-16728 | ||
Thermo Fisher Scientific, Waltham, MA | 22-8425-71 | ||
CellProfiler | Used for fluorescence image analysis | ||
Nikon, Japan | 10x objective | ||
American Type Culture Collection (ATCC), Manassas, VA | PCS-800-011 | ||
Ramé-Hart Instrument Co. (Netcong, NJ) | p/n 200-U1 | ||
Fisher, Canada | |||
Vitrocom, NJ, USA | 5015 and 5010 | Borosilicate capilary tube | |
(http://definetherain.org.uk/) | |||
Hamamatsu, Japan | LC-L1V5 | DEL UV light source | |
Dolomite | 3200063 | Disposable fluidic tubing | |
Dolomite | 3200302 | Disposable fluidic tubing | |
IDT, Coralville, IA | |||
Nikon, Melville, NY | Upright light microscope | ||
Cytec Industries, Woodland Park, NJ | |||
EV Group, Schärding, Austria | |||
Zymo Research, Irvine, CA | D5030 | ||
Photron, San Diego, CA | |||
IDT, Coralville, IA | |||
Gersteltec, Pully, Switzerland | SU-8 photoresist | ||
Fineline Imaging, Colorado Springs, CO | |||
Qiagen, Hilden, Germany | 203603 | ||
Image J | Used to assess droplet diameter | ||
Anachemia, Montreal, QC | |||
Excelitas, MA, USA | Broad-spectrum LED fluorescent lamp | ||
Galenvs Sciences Inc., Montreal, QC | DE1010 | ||
Hexpol TPE, Ă…mĂĄl, Sweden | Thermoplastic elastomer (TPE) | ||
Thermo Fisher Scientific, Waltham, MA | 13-400-518 | ||
Nikon, Japan | Used for image acquisition | ||
3M, St Paul, MN | Carrier Oil | ||
Thermo Fisher Scientific, Waltham, MA | R37605 | Blue fluorescent live cell stain (DAPI) | |
IDEX Health & Science, Oak Harbor, WA | P-881 | PEEK fittings | |
Sigma-Aldrich, Oakville, ON | 806552 | ||
Dow Corning, Midland, MI | |||
ThinkyUSA, CA, USA | ARV 310 | ||
Ihc world, Maryland, USA | IW-125-0 | ||
Zinsser NA, Northridge, CA | 2607808 | ||
Cetoni GmbH, Korbussen, Germany | |||
Sigma-Aldrich, Oakville, ON | 484431 | ||
Bio-Rad, Mississauga, ON | 1861096 | ||
Hitachi High-Technologies, Mississauga, ON | |||
Nikon, Melville, NY | Inverted microscope | ||
Nikon, Japan | |||
Loctite | AA 352 |
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