Name: measuring single-cell mitochondrial dna copy number and heteroplasmy using digital droplet polymerase chain reaction
Uploaded: 2022-07-12T13:30:00
Description: Watch this Scientific Journal Video about Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction at JoVE.com

Thank you to Dr. L Bozhilova for advice on the statistical analysis of droplet generation PCR data. Thank you to Dr. H Zhang for providing the oocytes used to generate data in Figure&#160;3C and Figure&#160;4B. This work was carried out by SPB at the Medical Research Council Mitochondrial Biology Unit (MC_UU_00015/9), University of Cambridge, and funded by&#160;a Wellcome Trust Principal Research Fellowship held by PFC (212219/Z/18/Z).

All experiments followed the ARRIVE guidelines and were approved by the University of Cambridge Animal Welfare Ethical Review Body (AWERB).
NOTE: All sample preparation steps prior to droplet generation must be performed in a clean pre-PCR work area, ideally in a UV-sterilized cabinet where possible. The protocol described here uses specific droplet generation PCR equipment (see Table of Materials), and while the general method should be applicable to other systems, it is reco...

<ol>
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The protocol described here is applicable across a wide range of cell types and species in addition to those discussed above, although careful optimization of new assay designs will be key to ensure that accuracy and repeatability of the method are maintained when moving away from previously validated primer/probe combinations. When working with single cells it is vital to ensure that sample collection is performed as accurately as possible (e.g., using stringent single-cell parameters when sorting cells by FACS) to ensu...

Mammalian mitochondrial (mt)DNA is a small (approx. 16.5 Kb), circular DNA genome residing in the mitochondrial matrix that encodes 37 genes, comprising two rRNAs, 22 tRNAs, and 13 protein-coding genes1. Unlike the nuclear genome, which contains one (haploid) or two (diploid) copies of each gene per cell, mtDNA is present in multiple copies in the mitochondria of each cell, ranging from tens of copies (e.g., mature spermatocytes) to hundreds of thousands of copies (e.g., oocytes)2,3. A consequence of this multi-copy nature is that mutations in the mtDNA genome, which may exist as single-nucleotide polymorphisms (SNPs), deletions, or duplications, can be present at varying levels in any given cell, making up anywhere from 0% to 100% of the cell's total mtDNA population. The existence of wild-type and mutant mtDNA genomes in the same cell is termed heteroplasmy, and pathogenic heteroplasmic mtDNA mutations are a major cause of mitochondrial disease, with several common neurological syndromes linked to underlying heteroplasmic mtDNA mutations4.
Two key parameters that contribute to the likelihood of a heteroplasmic mtDNA mutation causing clinical disease are the heteroplasmy level and the mtDNA copy number. Many heteroplasmic mutations display a threshold effect, with biochemical and clinical phenotypes only becoming apparent above a certain heteroplasmy level, typically around 80%5, and subsequently worsening as heteroplasmy increases further4. However, it is also important to consider the number of copies of mtDNA present in the cell, as this will influence the number of wild-type (i.e., 'healthy') mtDNA genomes that are present at a given heteroplasmy level. Studies in mitochondrial disease patients have highlighted the importance of this interplay between heteroplasmy and copy number5,6, and Filograna et al. recently reported an increase in mtDNA copy number alleviating symptoms despite unchanged heteroplasmy in a mouse model of mitochondrial disease7.
Whilst much has been done in recent years to improve the understanding of the pathogenesis and transmission of diseases caused by mtDNA heteroplasmy, most of this work has been conducted at the level of tissues rather than cells, comparing mean tissue heteroplasmy levels and copy number measurements acquired from bulk tissue biopsies and blood samples. Some well-established techniques, such as laser capture microdissection, allow for such measurements at the cellular level8,9; however, the recent explosion of high-throughput single-cell analysis methods, or so-called "Single-cell Omics"10, has created a requirement for methods that can accurately measure these crucial mtDNA parameters at the single-cell level.
The droplet generation PCR method takes advantage of recent advances in microfluidic technology to improve upon existing methods of quantitating unknown DNA concentrations via PCR amplification of specific target amplicons in the sample DNA11. Unlike qPCR, where the sample DNA is amplified in a single reaction, with the relative rate of accumulation of PCR product acting as the readout, this method splits the initial sample into thousands of individual droplets, thus compartmentalizing the sample DNA molecules into spatially separate reactions12. The subsequent PCR reaction proceeds in each individual droplet, with the PCR product only accumulating in droplets that contain the target DNA. The result of this reaction is a digital output in the form of a pool of droplets that either contain a copy of the target DNA and amplified PCR product, or do not contain target DNA. Using fluorescent DNA probes or a double-stranded (ds)DNA binding dye, the droplets containing amplified product can be counted, and the ratio of 'positive' to 'negative' droplets can be used to calculate the absolute number of DNA copies that were present in the initial sample. This absolute measurement, as opposed to the relative measurement acquired from a qPCR reaction, is the key factor that allows this methodology to be used for accurate measurement of mtDNA copy number and heteroplasmy in single cells11, and several recent studies have already utilized droplet generation PCR technology for this purpose13,14. This article presents a method for measuring mtDNA copy number and deletion heteroplasmy in single cells from both human and mouse tissue.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >50% Tween-20 solution</td>
			<td >Novex</td>
			<td >3005</td>
			<td ></td>
		</tr>
		<tr >
			<td >Automated droplet-generating oil&#160;</td>
			<td >Bio Rad</td>
			<td>1864110</td>
			<td>Commercial oil formulation used to generate the oil/droplet emulsion (used in Protocol Step 4.1)</td>
		</tr>
		<tr >
			<td >C1000 PCR machine with deep-well block</td>
			<td >Bio Rad</td>
			<td>1851197</td>
			<td>PCR thermocycler equipped with a deep-well heating block, used for cell lysis (Protocol Step 2.1.2.) and PCR cycling (Protocol Step 5)</td>
		</tr>
		<tr >
			<td >Collection plate cooling block</td>
			<td >Bio Rad</td>
			<td>12002819</td>
			<td>Cooling block that keeps samples chilled during droplet generation (used in Protocol step 4.3)</td>
		</tr>
		<tr >
			<td >ddPCR 96-well plates&#160;</td>
			<td >Bio Rad</td>
			<td>12001925</td>
			<td>96-well plates pipet tips designed for use in the QX200 AutoDG droplet generator, used for sample preparation (Protocol step 3.4) and droplet collection (Protocol step 4.3)</td>
		</tr>
		<tr >
			<td >ddPCR droplet reader oil&#160;</td>
			<td >Bio Rad</td>
			<td>1863004</td>
			<td>Commercial oil formulation used by the droplet reader (used in Protocol step 6.1)</td>
		</tr>
		<tr >
			<td >ddPCR Supermix for Probes (no dUTP)</td>
			<td >Bio Rad</td>
			<td>1863023</td>
			<td>Commercial supermix for use in ddPCR experiments utilising probes (used in Protocol Step 3.3)</td>
		</tr>
		<tr >
			<td >DG32 automated droplet generator cartridges&#160;</td>
			<td >Bio Rad</td>
			<td>1864108</td>
			<td>Microfluidic cartridges used in the QX200 AutoDG droplet generator to generate the oil/droplet emulsion (used in Protocol Step 4.3)</td>
		</tr>
		<tr >
			<td >Fetal bovine serum</td>
			<td >Gibco</td>
			<td >10270-106</td>
			<td>Qualified fetal bovine serum</td>
		</tr>
		<tr >
			<td >Foil plate covers&#160;</td>
			<td >Bio Rad</td>
			<td>1814040</td>
			<td>Foil plate covers used to seal droplet collection plates after droplet generation (used in Protocol step 4.6)</td>
		</tr>
		<tr >
			<td >HEK 293T cells</td>
			<td >Takara</td>
			<td >632180</td>
			<td>Commercial subclone of the transformed human embryonic kidney cell line, HEK 293, expressing the SV40 Large-T antigen</td>
		</tr>
		<tr >
			<td >HeLa cells</td>
			<td >ECACC</td>
			<td >93021013</td>
			<td>Human cervix epitheloid carcinoma cells</td>
		</tr>
		<tr >
			<td >High glucose DMEM</td>
			<td >Gibco</td>
			<td >13345364</td>
			<td>4.5g/L D-Glucose, with L-glutamine and sodium pyruvate</td>
		</tr>
		<tr >
			<td >Human cybrids</td>
			<td colspan="2">University of Miami</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse embryonic fibroblasts&#160;</td>
			<td colspan="2">Newcastle University</td>
			<td>Immortalized from C57Bl/6 mice</td>
		</tr>
		<tr >
			<td >Nuclease-free water</td>
			<td >Ambion</td>
			<td >AM9937</td>
			<td></td>
		</tr>
		<tr >
			<td >PCR plate seals</td>
			<td >Pierce</td>
			<td >SP-0027</td>
			<td>Clear adhesive plate seals, only used pre-droplet generation (foil seal must be used in step 4.6)</td>
		</tr>
		<tr >
			<td >Pipet Tip Waste Bins</td>
			<td >Bio Rad</td>
			<td>1864125</td>
			<td>Disposable collection bin used to collect discarded tips in the QX200 AutoDG droplet generator (used in Protocol step 4.3)</td>
		</tr>
		<tr >
			<td >Pipet tips for AutoDG system&#160;</td>
			<td >Bio Rad</td>
			<td>1864120</td>
			<td>Filtered pipet tips designed for use in the QX200 AutoDG droplet generator (used in Protocol step 4.3)</td>
		</tr>
		<tr >
			<td >Primary human dermal fibroblast cells</td>
			<td>Newcastle Biobank</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Primers/Probes</td>
			<td >IDT</td>
			<td >N/A</td>
			<td>Exact primer/probe sequences will be assay dependent. Primers and probes used in this study are given in Table 1</td>
		</tr>
		<tr >
			<td >Proteinase K 20 mg/mL solution</td>
			<td >Ambion</td>
			<td>AM2546</td>
			<td></td>
		</tr>
		<tr >
			<td >PX1 PCR plate sealer</td>
			<td >Bio Rad</td>
			<td>1814000</td>
			<td>Applies foil seals to ddPCR sample plates after droplet generation (used in Protocol Step 4.6)</td>
		</tr>
		<tr >
			<td >QX Manager software</td>
			<td >Bio Rad</td>
			<td>12012172</td>
			<td>Droplet reader set up &#38; analysis software (used in Protocol Steps 6 &#38; 7)</td>
		</tr>
		<tr >
			<td >QX200 AutoDG droplet generator</td>
			<td >Bio Rad</td>
			<td>1864101</td>
			<td>Automated microfluidic droplet generator (used in Protocol Step 4)</td>
		</tr>
		<tr >
			<td >QX200 droplet reader</td>
			<td >Bio Rad</td>
			<td>1864003</td>
			<td>Droplet reader (used in Protocol Step 6)</td>
		</tr>
		<tr >
			<td >Trizma pre-set crystals pH 8.3</td>
			<td >Sigma</td>
			<td >T8943-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Following droplet generation, a clear layer of opaque droplets is visible floating on top of the oil phase in each well (Figure 1B). Droplet formation can be adversely affected by the presence of detergents in the input lysate when performing experiments on single cells. Using the lysis protocol described in 2.1.2., droplet yields above the recommended level of 10,000 are routinely achieved, despite the presence of a small residual amount of TWEEN-20 in the final sample (<strong class="xfig"...

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

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 ...

This protocol can be used to accurately measure the number of copies of mitochondrial DNA present in individual cells and the level of certain types of mitochondrial DNA mutation. Previous methods can accurately make these measurements in tissue samples containing many cells. The main advantage of this technique is that we can make the same measurements in single cells.This method is of particularly use to those studying mitochondria and will have wide applications in this field. Effective isolation and lysis of single cells is key to this technique. Previous experience with single cell sorting would be very helpful.To begin, prepare the master mix for the PCR without the sample DNA on ice, as described in the text. After vortexing briefly, distribute the required volume of the prepared master mix to each well of a droplet generation PCR 96-well plate, arranging the samples in full columns. Next, add the master mix to any empty well in incomplete columns for using them as non-template controls.Then add the input DNA to each well to make the total volume of each well 22 microliters. Seal the plate with an adhesive plate seal and place it on a shaker at 2000 RPM for one minute. Then centrifuge the plate at 1000 times G for one minute at four degrees Celsius before placing it on ice.For droplet generation, first ensure that the droplet generator is powered on and check whether a bottle of droplet generating oil is loaded to position E of the instrument deck. Then click Configure Sample Plate on the touch screen. After selecting all columns on the sample plate containing the samples or blanks, click on Okay to confirm the plate configuration.Next, load the droplet generator cartridges to position B on the instrument deck so that all lights turn green and place an empty tip waste trough into position C.Then load filter tip boxes with lids removed to position D on the instrument stage so that all lights turn green. Next, remove the adhesive seal before loading the sample plate to position F of the instrument deck so that the light turns green. Also load an empty droplet collection 96-well plate placed into a cool block, pre-chilled at minus 20 degrees Celsius, at position G so that the light turns green.Click Start Droplet Generation on the instrument touch screen and the droplet generator lid will close automatically. Once droplet generation is complete, check the sample collection plate visually to confirm that a droplet emulsion layer is present above the oil phase in each well. Next, power on the plate sealer and set the temperature to 180 degrees Celsius and seal time to five seconds.Allow the machine to reach the operating temperature. Then place the sample collection plate on the plate holder of the plate sealer and place a fresh foil seal on the top of the plate with the red line facing upwards. When the plate sealer has reached its operating temperature, press eject on the instrument touch screen.After placing the plate holder in the drawer of the plate sealer, press seal to close the drawer. Once the sealing is complete, the drawer will automatically open. Remove the sealed plate and place it on the cold block.Then turn off the plate sealer. For running the PCR, place the sealed droplet collection plate in a PCR machine with a 96 deep-well block. After the PCR is complete, power on the droplet reader and the connected computer.Load the plate with the post-PCR samples into the droplet reader plate holder. While keeping the foil lid on the sample plate, place the cover on the top of the holder and secure it in place with black locking clips. Next, to open the droplet reader lid, press the open/close button.Then load the droplet reader plate holder with the sample plate into the reader chamber and place it securely on the magnetic base. Press the open/close button again to close the lid. Open the analysis software interface.Select the Add Plate tab, click Add Plate, and then Configure Plate. In the Plate Information tab, enter a plate name, select Supermix for Probes, No DUTP from the Supermix Dropdown menu, and enter a file name under Save Data File As.In the Well Selection tab, highlight the wells to be analyzed and click Include Selected Wells. Then in the Well Information tab, select the wells to be annotated.Select Direct Quantification from the Experiment Type dropdown menu. Then populate the sample description, sample type, and target name boxes, and add well notes and or plate notes as required. Then click Apply.Once the plate configuration is complete, click Start Run. For result analysis, select the Data Analysis tab and open the file to be analyzed from the My Data Files menu. Then click the 1D Amplitude tab for single-color experiments, or the 2D Amplitude tab for two-color experiments.Next, select the wells that require thresholding. Use the Threshold Line Mode tool to manually apply the threshold in the clear space between the positive and negative populations on the amplitude plot, ensuring that the crosshair is placed such that the four droplet populations are clearly separated. Once the threshold is applied for all samples, export the results as a csv file for further analysis by clicking on the Data Table tab, Import/Export, and selecting Export Visible Data to CSV.Enter a suitable file name and click Save. The PCR and droplet reading procedures used in this protocol demonstrated the presence of clearly-separated populations of positive and negative droplets, both in the fam and hex channels. Mitochondrial DNA populations positive for both the probes could also be distinguished from single cell lysates.This method also showed that for samples with deletions in the mitochondrial DNA, a mixed population of double-positive droplets and droplets positive for only the non-deleted portion of the DNA are present. This method has been successfully used to count the mitochondrial DNA copy number per cell in a variety of mammalian cells, including mouse oocytes and primary mouse embryonic cells. Furthermore, in cells with high deletion heteroplasmy, the population positive for a single probe was found to be significantly bigger than the double-positive population.However, in cells with low-deletion heteroplasmy, both populations were of similar size. So accurate results always ensure that the droplet generation has been successful and that thresholding has been appropriately applied using the 1D or 2D amplitude view. Unused lysate can be used for other single cell assays, such as pyrosequencing, which can measure single nucleotide polymorphism heteroplasmy.The development of this technique has allowed us to make accurate measurements of mitochondrial DNA copy number, and deletion heteroplasmy in individual cells, which was previously not possible.

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