Name: methylation specific multiplex droplet pcr using polymer droplet generator device for hematological diagnostics
Uploaded: 2020-06-29T14:00:00.000+00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics at JoVE.com

The authors acknowledge financial support from the National Research Council of Canada.

<ol>
	<li>Teitell, M., Richardson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+the+immune+system.">DNA methylation in the immune system.</a> Clinical Immunology. 109 (1), 2-5 (2003).</li><li>Suarez-Alvarez, B., Rodriguez, R. M., Fraga, M. F., L&#243;pez-Larrea, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+A+promising+landscape+for+immune+system-related+diseases.">DNA methylation: A promising landscape for immune system-related diseases.</a> Trends in Genetics. 28 (10), 506-514 (2012).</li><li>Su&#225;rez-&#193;lvarez, B., Raneros, A. 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C., Veres, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoplastic+elastomers+for+microfluidics:+Towards+a+high-throughput+fabrication+method+of+multilayered+microfluidic+devices.">Thermoplastic elastomers for microfluidics: Towards a high-throughput fabrication method of multilayered microfluidic devices.</a> Lab on a Chip. 11 (18), 3193-3196 (2011).</li><li>Roy, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cellular+lysis+to+microarray+detection,+an+integrated+thermoplastic+elastomer+(TPE)+point+of+care+Lab+on+a+Disc.">From cellular lysis to microarray detection, an integrated thermoplastic elastomer (TPE) point of care Lab on a Disc.</a> Lab on a Chip. 15 (2), 406-416 (2015).</li><li>Malic, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+subtyping+of+white+blood+cells+using+a+thermoplastic+elastomer-based+microfluidic+emulsification+device+for+multiplexed,+methylation-specific+digital+droplet+PCR.">Epigenetic subtyping of white blood cells using a thermoplastic elastomer-based microfluidic emulsification device for multiplexed, methylation-specific digital droplet PCR.</a> Analyst. 44 (22), 6541-6553 (2019).</li><li>Malic, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-based+microfluidic+chip+for+rapid+and+efficient+immunomagnetic+capture+and+release+of+Listeria+monocytogenes.">Polymer-based microfluidic chip for rapid and efficient immunomagnetic capture and release of Listeria monocytogenes.</a> Lab Chip. 15 (20), 3994-4007 (2015).</li><li>Malic, L., Morton, K., Clime, L., Veres, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-thermoplastic+nanoplasmonic+microfluidic+device+for+transmission+SPR+biosensing.">All-thermoplastic nanoplasmonic microfluidic device for transmission SPR biosensing.</a> Lab Chip. 13 (5), 798-810 (2013).</li></ol>

There are no conflicts to declare.

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 manufacturing9,10,11,12,13. In addition, it is also optically clear and exhibits low fluorescence background in the visible spectrum which is an attractive feature for future integration of a complete sample-to-answer workflow using mdPCR. The PCR reagent recipes are also provided in a homebrew format to allow for facile multiplex gene quantification, while also maintaining stability throughout the thermal cycling protocol. As such one of the advantages of the presented devices and protocols is the flexibility in customizing the device design and reagents used for a particular application, which is difficult to achieve with commercial products and proprietary formulations. Furthermore, it removes the necessity of expensive instrumentation to perform the experiment.
Appropriate thermoplastic material selection of the microfluidic droplet generator is a critical parameter that can circumvent cumbersome surface treatment to render the device hydrophobic for efficient droplet formation. In addition, the selection of an optimized mdPCR buffer is also critical for maintaining droplet stability through the thermal cycling process &#8211; whilst not compromising the PCR efficiency and hydrolysis probe functionality. Further modification and optimization of the mdPCR buffer is, therefore, encouraged to arrive at highly specific and sensitive PCR amplification of selected gene targets, coupled with fluorescence based probe detection. This serves to increase signal-to-noise ratios, reducing the instances of &#8216;rain&#8217; and simplifying the experimental analysis and identification of positive droplet populations. Troubleshooting of the experiment depends on assessing the critical steps from thermoplastic material selection to polymerase concentration to adapting the temperature ramp in the thermal cycling program in order to ensure droplet stability.
The current limitation of the described protocol is that it still necessitates manual sample transfer from the droplet generator to the thermal cycler and finally to a droplet imaging device and instrument such as a fluorescence microscope. Future efforts however are directed at the miniaturization of mdPCR and integration of these steps whereby droplet generation, PCR and imaging can be performed on a single automated platform.
The obtained results in Figure 6 demonstrate the versatility of this mdPCR approach for performing differential white blood cell quantification. The proposed method is more precise and reproducible than immunofluorescence imaging, which is often affected by user manipulation, giving rise to unwanted measurement errors. This is also the case for flow cytometry techniques that also rely on an immune-based method of detection which is highly dependent on cell viability, as well as multiple protocol steps prone to user error. Alternative methods such as real-time quantitative PCR (qPCR) for the quantification are often adversely affected by low copy number gene targets, a drawback that is remedied through mdPCR without the need for a dedicated calibration curve11. The presented mdPCR approach, therefore, presents a unique molecular approach to white blood cell differential counts that is based on methylation profiles of specific gene targets. Calibration curves for individual gene targets are also not necessary since mdPCR is based on an absolute quantification of binary positive and negative signals using Poisson distribution for CPD calculation.
The precise quantification of non-methylated gene islands, especially of low copy number genes such as FOXP3, presents a multitude of opportunities for clinical diagnosis. This is highlighted by the capacity of such an approach to identify known methylation patterns correlated to disease onset and progression. mdPCR, as a molecular approach with absolute quantification can be employed beyond methylation studies and can be applied to appropriately preserved samples, therefore removing the dependence on fresh clinical samples, and further expanding its applications. Future automation and miniaturization of this technique will be of great value for rapid and accurate 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 samples cannot be frozen for shipment or later analysis.
Epigenetic markers have recently emerged as powerful analytical tools for the study of phenotypic variations. Subsequently, human leukocyte populations have been shown to have cell-lineage DNA methylation patterns that allow for the precise characterization of WBC subsets. Subtyping based on epigenetic markers provides a promising alternative that does not depend on fresh blood sample collection or expensive antibodies and can be exploited as a biomarker for disease onset and susceptibility1,2,3,4,5.
Genome-wide studies have been performed for extensive mapping of methylated specific CG-rich regions in the genome (CpG islands) in leukocyte subtypes to identify candidate epigenetic markers specific to leukocyte subtypes. PCR protocols have been developed because of this reason for methylated gene regions, e.g., CD3Z and FOXP3, corresponding to CD3+ T-Cells and CD4+ CD25+ Regulatory T-Cells (T-Regs), respectively. Wiencke et al. have demonstrated the utility of duplex droplet PCR for epigenetic subtyping of T-Cells, yielding results that highly correlate with flow activated cell sorting (FACS) analysis6. This quantitative genetic analysis method relies on partitioning the template nucleic acid molecules and PCR reagents into thousands of discrete, volumetrically defined, sub-nanoliter sized droplets containing zero, one or more target nucleic acid copies, using water-in-oil emulsions enabled by microfluidics7,8. The PCR amplification is performed within each individual droplet and the endpoint fluorescence intensity of each droplet is measured, allowing absolute quantification of targets present in the sample. Droplet PCR has been established to be more precise, accurate, and technically simpler than standard qPCR, making it a more favorable DNA methylation-based method for clinical evaluation of T-Cells. Although a rapidly emerging subtyping methodology, multiplexed epigenetic analysis to probe for various methylated CpG regions simultaneously is lacking. This is necessary for routine leukocyte differential counts.
Herein, a thermoplastic elastomer (TPE) droplet microfluidic device is presented and employed for methylation-specific multiplex droplet PCR (mdPCR). The technology has been used to delineate specific leukocyte subtypes, CD3+ T-Cells and CD4+ CD25+ T-Regs, based on cell-lineage DNA methylation patterns, i.e., epigenetic variation of CD3Z and FOXP3 CpG regions, respectively. A detailed protocol for DNA extraction, bisulfite conversion and mdPCR is described in concert with a fabrication method for a TPE droplet generation device. Representative results of the method are compared to those of immunofluorescence staining highlighting the utility of the proposed approach.

<table><tbody><tr><td>Bio-Rad, Mississauga, ON</td><td>TFI0201</td><td>PCR tube</td><td></td></tr><tr><td>RAN Biotechnologies, Beverly, MA</td><td>008-FluoroSurfactant</td><td>Fluoro-surfactant</td><td></td></tr><tr><td>Silicon Quest International, Santa Clara, CA</td><td></td><td></td><td></td></tr><tr><td>Oxford Instruments, Abingdon, UK</td><td></td><td>EMCCD camera</td><td></td></tr><tr><td>Thermo Fisher Scientific, Waltham, MA</td><td>MA5-16728</td><td></td><td></td></tr><tr><td>Thermo Fisher Scientific, Waltham, MA</td><td>22-8425-71</td><td></td><td></td></tr><tr><td>CellProfiler</td><td></td><td>Used for fluorescence image analysis</td><td></td></tr><tr><td>Nikon, Japan</td><td></td><td>10x objective</td><td></td></tr><tr><td>American Type Culture Collection (ATCC), Manassas, VA</td><td>PCS-800-011</td><td></td><td></td></tr><tr><td>Ram&amp;eacute;-Hart Instrument Co. (Netcong, NJ)</td><td>p/n 200-U1</td><td></td><td></td></tr><tr><td>Fisher, Canada</td><td></td><td></td><td></td></tr><tr><td>Vitrocom, NJ, USA</td><td>5015 and 5010</td><td>Borosilicate capilary tube</td><td></td></tr><tr><td>(http://definetherain.org.uk/)</td><td></td><td></td><td></td></tr><tr><td>Hamamatsu, Japan</td><td>LC-L1V5</td><td>DEL UV light source</td><td></td></tr><tr><td>Dolomite</td><td>3200063</td><td>Disposable fluidic tubing</td><td></td></tr><tr><td>Dolomite</td><td>3200302</td><td>Disposable fluidic tubing</td><td></td></tr><tr><td>IDT, Coralville, IA</td><td></td><td></td><td></td></tr><tr><td>Nikon, Melville, NY</td><td></td><td>Upright light microscope</td><td></td></tr><tr><td>Cytec Industries, Woodland Park, NJ</td><td></td><td></td><td></td></tr><tr><td>EV Group, Sch&amp;auml;rding, Austria</td><td></td><td></td><td></td></tr><tr><td>Zymo Research, Irvine, CA</td><td>D5030</td><td></td><td></td></tr><tr><td>Photron, San Diego, CA</td><td></td><td></td><td></td></tr><tr><td>IDT, Coralville, IA</td><td></td><td></td><td></td></tr><tr><td>Gersteltec, Pully, Switzerland</td><td></td><td>SU-8 photoresist</td><td></td></tr><tr><td>Fineline Imaging, Colorado Springs, CO</td><td></td><td></td><td></td></tr><tr><td>Qiagen, Hilden, Germany</td><td>203603</td><td></td><td></td></tr><tr><td>Image J</td><td></td><td>Used to assess droplet diameter</td><td></td></tr><tr><td>Anachemia, Montreal, QC</td><td></td><td></td><td></td></tr><tr><td>Excelitas, MA, USA</td><td></td><td>Broad-spectrum LED fluorescent lamp</td><td></td></tr><tr><td>Galenvs Sciences Inc., Montreal, QC</td><td>DE1010</td><td></td><td></td></tr><tr><td>Hexpol TPE, &amp;Aring;m&amp;aring;l, Sweden</td><td></td><td>Thermoplastic elastomer (TPE)</td><td></td></tr><tr><td>Thermo Fisher Scientific, Waltham, MA</td><td>13-400-518</td><td></td><td></td></tr><tr><td>Nikon, Japan</td><td></td><td>Used for image acquisition</td><td></td></tr><tr><td>3M, St Paul, MN</td><td></td><td>Carrier Oil</td><td></td></tr><tr><td>Thermo Fisher Scientific, Waltham, MA</td><td>R37605</td><td>Blue fluorescent live cell stain (DAPI)</td><td></td></tr><tr><td>IDEX Health &amp;amp; Science, Oak Harbor, WA</td><td>P-881</td><td>PEEK fittings</td><td></td></tr><tr><td>Sigma-Aldrich, Oakville, ON</td><td>806552</td><td></td><td></td></tr><tr><td>Dow Corning, Midland, MI</td><td></td><td></td><td></td></tr><tr><td>ThinkyUSA, CA, USA</td><td>ARV 310</td><td></td><td></td></tr><tr><td>Ihc world, Maryland, USA</td><td>IW-125-0</td><td></td><td></td></tr><tr><td>Zinsser NA, Northridge, CA</td><td>2607808</td><td></td><td></td></tr><tr><td>Cetoni GmbH, Korbussen, Germany</td><td></td><td></td><td></td></tr><tr><td>Sigma-Aldrich, Oakville, ON</td><td>484431</td><td></td><td></td></tr><tr><td>Bio-Rad, Mississauga, ON</td><td>1861096</td><td></td><td></td></tr><tr><td>Hitachi High-Technologies, Mississauga, ON</td><td></td><td></td><td></td></tr><tr><td>Nikon, Melville, NY</td><td></td><td>Inverted microscope</td><td></td></tr><tr><td>Nikon, Japan</td><td></td><td></td><td></td></tr><tr><td>Loctite</td><td>AA 352</td><td></td><td></td></tr></tbody></table>

methylation specific multiplex droplet pcr using polymer droplet generator device for hematological diagnostics

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 WBC&#160;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.

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 subsequent thermoforming of thermoplastics using hot embossing. Once the epoxy mold was obtained, thermoplastic elastomer was embossed. Following the embossing, the TPE was demolded, and devices were cut. A flat TPE substrate was used to seal the device. Holes were punched in the top cover, and the two substrates were bonded. Finally, the necessary tubing for world-to-chip connections were inserted and the device was ready for use. Sample images of fabricated master mold, TPE material and embossed devices are shown in Figure 2. Assembled device with tubing interconnects was operated with a pair of independent programmable syringe pumps to generate emulsions for mdPCR. Methylation-specific mdPCR was performed using bisulfite treated DNA extracted from frozen PBMCs. Appropriate primers and hydrolysis probes for CD3Z, FOXP3, and C-Less genes were added to the PCR mix for emulsification. Following droplet generation of appropriate size (approximately 72 &#956;m diameter), the emulsion was subjected to a thermal cycling protocol. Finally, the droplets were introduced into a glass capillary having 1 mm width and 50 &#181;m height for imaging. This results in a monolayer distribution of droplets ideal for fluorescence image acquisition (Figure 3). Images were recorded for each of the four wavelengths. An image analysis was then performed to identify all droplets (Figure 4) meeting the threshold criteria established through &#8216;definetherain&#8217; algorithm. The fluorescence intensity of all droplets in their respective fluorophore is then plotted and the threshold of positive and negative droplets was established (Figure 5). Subsequently, identification and counting of these droplets were performed which were above the selected threshold. The CPD values were then calculated and the percentage of CD3+ T-Cell and CD4+ 25+ T-Regs was established based on methylated CD3Z and FOXP3 copies, respectively, with respect to the CPD of total cells, or C-LESS gene (Figure 6). The percent values were then compared to those obtained through immunofluorescence imaging of T-Cell and T-Reg populations using appropriate antibodies.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FOXP3 Forward</td>
			<td colspan="2">GGG TTT TGT TGT TAT AGT TTT TG</td>
		</tr>
		<tr>
			<td>FOXP3 Reverse</td>
			<td colspan="2">TTC TCT TCC TCC ATA ATA TCA</td>
		</tr>
		<tr>
			<td>CD3Z Forward</td>
			<td colspan="2">GGA TGG TTG TGG TGA AAA GTG</td>
		</tr>
		<tr>
			<td>CD3Z Reverse</td>
			<td colspan="2">CAA AAA CTC CTT TTC TCC TAA CCA</td>
		</tr>
		<tr>
			<td>C-LESS Forward</td>
			<td colspan="2">TTG TAT GTA TGT GAG TGT GGG AGA GA</td>
		</tr>
		<tr>
			<td>C-LESS Reverse</td>
			<td colspan="2">TTT CTT CCA CCC CTT CTC TTC C</td>
		</tr>
		<tr>
			<td>FOXP3 Probe</td>
			<td colspan="2">/56-FAM/CA ACA CAT C/ZEN/C AAC CAC CAT /3IABkFQ/</td>
		</tr>
		<tr>
			<td>CDZ3 Probe</td>
			<td colspan="2">/56-HEX/CC AAC CAC C/ZEN/A CTA CCT CAA /3IABkFQ/</td>
		</tr>
		<tr>
			<td>C-Less Probe</td>
			<td colspan="2">/56-CY5/CT CCC CCT C/ZEN/T AAC TCT AT/3IABkFQ/</td>
		</tr>
	</tbody>
</table>
Table 1: Primer and hydrolysis probe design.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Stock Solution</td>
			<td>Master Mix Volume</td>
			<td>Working Concentration</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>1 M</td>
			<td>2 &#181;L</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>1 M</td>
			<td>10 &#181;L</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>25 mM</td>
			<td>16 &#181;L</td>
			<td>4 mM</td>
		</tr>
		<tr>
			<td>C-LESS Primers</td>
			<td>10 &#181;M</td>
			<td>10 &#181;L</td>
			<td>1 &#181;M each</td>
		</tr>
		<tr>
			<td>CD3Z Primers</td>
			<td>10 &#181;M</td>
			<td>10 &#181;L</td>
			<td>1 &#181;M each</td>
		</tr>
		<tr>
			<td>FOXP3 Primers</td>
			<td>10 &#181;M</td>
			<td>10 &#181;L</td>
			<td>1 &#181;M each</td>
		</tr>
		<tr>
			<td>C-LESS probe</td>
			<td>10 &#181;M</td>
			<td>5 &#181;L</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>CD3Z probe</td>
			<td>10 &#181;M</td>
			<td>5 &#181;L</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>FOXP3 probe</td>
			<td>10 &#181;M</td>
			<td>5 &#181;L</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>HotStarTaq DNA Polymerase</td>
			<td>5 Units/&#181;L</td>
			<td>8 &#181;L</td>
			<td>0.4 Units/&#181;L</td>
		</tr>
		<tr>
			<td>Bisulfite-treated DNA Target</td>
			<td>Variable</td>
			<td>Variable</td>
			<td>Variable</td>
		</tr>
		<tr>
			<td>Nuclease-Free Water</td>
			<td></td>
			<td>Variable</td>
			<td></td>
		</tr>
		<tr>
			<td>Total Volume</td>
			<td></td>
			<td>100 &#181;L</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Master mix recipe for mdPCR.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Optical configuration</td>
			<td>Exposure</td>
			<td>DIA lamp</td>
			<td>Florescent light</td>
		</tr>
		<tr>
			<td>Bright field</td>
			<td>50 ms</td>
			<td>ON</td>
			<td>OFF</td>
		</tr>
		<tr>
			<td>FAM</td>
			<td>300 ms</td>
			<td>OFF</td>
			<td>20%</td>
		</tr>
		<tr>
			<td>HEX</td>
			<td>300 ms</td>
			<td>OFF</td>
			<td>20%</td>
		</tr>
		<tr>
			<td>Cy5</td>
			<td>400 ms</td>
			<td>OFF</td>
			<td>20%</td>
		</tr>
	</tbody>
</table>
Table 3: Settings for optical configuration and image acquisition.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61421/61421fig1v2.jpg" /> 
Figure 1: Rapid prototyping of the droplet generator.&#160;Schematic illustration of the process used for the fabrication of (A) master mold and (B) TPE-based microfluidic emulsification device. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61421/61421fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61421/61421fig2v2.jpg" /> 
Figure 2: Components involved in the fabrication process.&#160;(A) Silicon master, (B) PDMS replica, (C) epoxy mold, (D) TPE material extruded into sheets and packaged in rolls, (E) embossed TPE and (D) assembled device. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61421/61421fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61421/61421fig3v2.jpg" /> 
Figure 3: Fluorescence image acquisition of droplets following thermal cycling.&#160;(A) Brightfield image of the droplets in a capillary that allowed monolayer image acquisition. (B) Cy5 filter image of C-LESS gene targets representing total cell count. (C) HEX filter image of methylated CD3Z gene target correlated to CD3+ T-Cell count. (D) FAM filter image of methylated FOXP3 gene target correlated to CD4+ CD25+ T-Reg count. Scale bar is 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61421/61421fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61421/61421fig4v2.jpg" /> 
Figure 4: Pipeline used for identifying droplets that meet threshold intensity.&#160;Images were first organized into appropriate fluorescence filters using file naming. The images were then converted to grayscale and relative intensities rescaled based on the minimum and maximum droplet fluorescence intensities. The threshold was then selected according to values obtained from &#8216;definetherain&#8217; algorithm and the objects &#8211; or droplets &#8211; meeting the defined criteria were identified and quantified. Finally, the objects were filtered according to eccentricity and size to obtain the refined droplet count of spherical objects meeting 75 &#181;m diameter size. The quantified objects and their intensities were then exported to a spreadsheet software for downstream analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61421/61421fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61421/61421fig5v2.jpg" /> 
Figure 5: Intensity scatter plots for fluorescence intensity of droplets following mdPCR.&#160;(A) C-LESS gene amplification with Cy5 hydrolysis probe representing the total cell count as the target region was devoid of cytosine residues and, therefore, resistant to bisulfite treatment. (B) Methylated CD3Z gene amplification with HEX hydrolysis probe, indicative of CD3+ T-Cell population. (C) Methylated FOXP3 gene amplification with FAM hydrolysis probe, representing the CD4+ CD25+ T-Reg population. All the scatter plots were subjected to &#8216;definetherain&#8217; algorithm to set the appropriate threshold whereby the positives were within 3 standard deviations of the positive cluster mean. The amount of &#8216;rain&#8217; was also established so that it does exceed 1% of the positive defined droplets. Each dataset for analysis consisted of ~5,000 droplets and was run in triplicate (see Supplementary Information for raw data and analysis). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61421/61421fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61421/61421fig6v2.jpg" /> 
Figure 6: CPD values of all gene targets and determination of white blood cell subset percentage.&#160;(A) The calculated CPD values based on Poisson distribution of the positive droplet count as a ratio of total droplets. The input represents the theoretical CPD expected based on 740 ng of bisulfite treated DNA, 75 &#181;m diameter droplets, assuming 6.6 pg for 1 gene copy. The input theoretical CPD was 0.25 and correlated with the C-LESS CPD representing the total cell count. (B) The ratio of CPD for CD3Z and FOXP3 with respect to C-LESS was then used to obtain the percent of CD3+ T-Cells and CD4+ CD25+ T-Regs, respectively. These ratios as percent of total leukocytes were then compared with values obtained by immunofluorescence imaging. As demonstrated, the values from mdPCR and immunofluorescence analysis correlate with no significant difference, with standard deviation errors from mdPCR being much less pronounced. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61421/61421fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Information. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61421/Supplemental_Information.zip" target="_blank">Please click here to download these files.</a>

Watch this Scientific Journal Video about Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics at JoVE.com

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

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, ...

methylation-specific-multiplex-droplet-pcr-using-polymer-droplet

Nanyang Technological University

Research

JoVE Journal

Bioengineering

5.1K Views.  National Research Council of Canada. Dit protocol presenteert een flexibele multiplex druppel PCR workflow die nauwkeurige differentiële leukocyten tellen op basis van epigenetische methylatie markers mogelijk maakt. Deze flexibiliteit kan de vertaling van mdPCR naar klinisch gebruik vergemakkelijken. Deze methode biedt resultaten die nauw opvragen bij degenen die zijn verkregen met behulp van immunofluorescente kleuringsmethoden.In tegenstelling tot immunofluorescente kleuring, echter, deze methode vereist geen verse bl...

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

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

Genetics

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Cancer Research

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron ...

Medicine

Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes

Source: Carballar-Lejaraz&#250;, R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62607">Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations.</a> J. Vis. Exp. (2021).
This video demonstrates the role of digital droplet polymerase chain reaction in identifying indel mutation in a DNA sample. The high precision and sensitivity of ddPCR enable accurate and consistent detection of rare mutations present in a sample, even at low levels. It can identify and quantify indel mutations in different regions of the genome, providing valuable insights into the mechanisms of mutation and the functional consequences of these mutations.&#160;

Source: Carballar-Lejaraz&#250;, R., et al. Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations. J. ...

JoVE Encyclopedia of Experiments

Biological Techniques

PCR Techniques

Multiplex Droplet Polymerase Chain Reaction to Detect DNA Methylation in Leukocytes

Source: Malic, L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61421">Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics.</a>&#160;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.

Source: Malic, L., et al. Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics.&#160;J. Vis. ...

Droplet Digital Polymerase Chain Reaction: A Method to Generate Nanodroplet PCR Reactions for Detecting Rare Tumor Mutations

Source: Bonner, E. R. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59721" target="_blank">Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids.</a> J. Vis. Exp. (2019)
In this video, we present a step-by-step procedure to generate emulsified nano-droplets to detect target nucleic acids by digital polymerase chain reaction or dPCR.

Source: Bonner, E. R. et al. Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids. J. Vis. Exp. (2019)
In this video, we ...

Cancers of the Nervous System

In vitro studies

Duplex Digital PCR for Simultaneous Quantification of Dual Genetic Markers

To begin this procedure, first, prepare 100 micromole per liter stock concentrations for all primers in molecular-grade water and probes in TE pH 8 buffer, as described in the protocol text. Next, prepare a master mix by mixing appropriate amounts of digital PCR mix, forward and reverse primers, the fluorescent probe, and nuclease-free water. Pipette up and down at least 10 times to mix, while taking caution not to introduce air bubbles in the solution.
Since dPCR master mix is more viscous than conventional qPCR master mix, it&#39;s necessary to use the mixing by pipetting technique in order to create a homogeneous solution. This allows for accurate dPCR quantification downstream.
To make the assay mixture for droplet generation for running samples in duplicate, pipette 36 microliters of the master mix into a regular PCR plate. To each of the 36-microliter master mixes, mix in 12 microliters of DNA template, leaving the corresponding replicate wells empty on the plate. Include positive controls to ensure the assay is running properly. Include No Template Controls or NTCs to ensure there is no contamination within the plate, and to set the fluorescent baseline later for data analysis.
Prior to setting up the droplet generator, mix the assay mixtures by using a multichannel pipette to pipette the mixtures up and down approximately 15 times. Ensure that the pipette tip stays within the liquid to avoid making excess bubbles within the mixture.
Next, insert cartridge one, containing eight wells, into a white cartridge holder and click the cartridge holder shut. Cartridge one is now firmly in place, and cannot be dislodged from the holder while generating droplets.
Using a multichannel pipette, gently transfer 20 microliters of the assay mixture into the middle position of the cartridge marked &#39;Sample&#39; without introducing air bubbles. Pipette in 70 microliters of droplet generation oil to the left side of the cartridge marked &#39;Oil.&#39;
Cover the cartridge with a gasket, making sure the gasket is flat and held evenly by the four ticks toward the edge of the cartridge. Press the green-lit button on the droplet generator to open the door and place the cartridge. Press the button again to close the generator. Once the door closes, the green button is dimmed and the door cannot be reopened.
Droplet generation will begin immediately and continue for approximately 1 minute.
While the droplet generation is in progress, place cartridge two in a second white cartridge holder, and prepare it in the same manner as for cartridge one. When the droplet generation is complete, the dimly-lit button will turn green.
Open the droplet generator door. Remove the white cartridge holder containing cartridge one and set it aside. Place cartridge two into the droplet generator.
Remove the gasket from cartridge one and discard. Do not unclick the white cartridge holder, as the action may break the newly-generated droplets.
Using a multichannel pipette set to 40 microliters, insert the tips into the third column of the cartridge marked droplets at a 45-degree angle, and slowly pipette up all the droplets. Transfer them to the final PCR plate by putting the pipette tip against the well wall approximately halfway down and expelling the droplet slowly.
In the same way, when the droplet generation for cartridge two is complete, remove the gasket from cartridge two, and transfer the generated droplets to the final PCR plate. Place a pierceable foil cover on top of the plate and place it on a plate sealer. Set the sealer to 180 degrees Celsius. Press &#34;Play&#34; on the sealer and seal for 10 seconds.
To begin this procedure, place the sealed final PCR plate in the thermal cycler. Use a thermal cycler that is compatible with the final PCR plate, and with a temperature ramping speed of 2 degrees Celsius per second.
Run the following thermal program: 10 minutes at 95 degrees Celsius followed by 40 cycles of 30 seconds at 94 degrees Celsius and 60 seconds at 60 degrees Celsius followed by a 10-minute hold at 98 degrees Celsius. Upon completion of cycling, transfer the plate to a droplet reader for automatic measurement of fluorescence in each droplet in each well.
Ensure the droplets are at room temperature before proceeding with droplet reading. Start by opening the accompanying software to set up the droplet reading. In the default &#34;Setup&#34; menu containing a schematic of an empty 96-well plate, double-click on well A1 to open the menu containing three sections -- &#34;Sample,&#34; &#34;Assay 1,&#34; and &#34;Assay 2.&#34;
In the sample section, type the sample ID into the box labeled &#34;Name&#34; and check the box to the right marked &#34;Apply.&#34; Next, click the dropdown menu labeled &#34;Experiment.&#34; Choose &#34;RED&#34; for Rare Event Detection and click &#39;Enter.&#39;
Move to the section denoted as &#34;A1.&#34; In the &#34;Name&#34; section, fill out the assay and click &#39;Enter.&#39; In the box below labeled &#34;Type,&#34; click the dropdown menu, choose &#34;Channel 1 Unknown,&#34; and click &#39;Enter.&#39;
Move to the section denoted as &#34;A2.&#34; In the &#34;Name&#34; section, fill out the assay and click &#39;Enter.&#39; In the box below labeled &#34;Type,&#34; click the dropdown menu, choose &#34;Channel 2 Unknown,&#34; and click &#39;Enter.&#39;
All the information from the previous steps is now present in well A1.
Name all subsequent wells containing droplets. To save total setup time, click &#39;Shift&#39; or &#39;Control&#39; to choose multiple wells simultaneously. When the digital depiction of the plate mirrors the physical plate, press &#34;OK&#34; at the bottom right of the menu. In the new menu that appears at the top of the plate schematic, under the &#34;Template&#34; section, choose &#34;Save As&#34; and name and save the plate.
To the left of the screen, click &#34;Run&#34; and select the appropriate Dye Set in the pop-out &#34;Run Options&#34; window. Data collection will initiate and is displayed in real-time time in the software.

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

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