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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Research

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Medicine

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of arthroplasty. Infection requires complex treatment, may result in long hospitalization and causes considerable costs. Multiple surgical revisions can be necessary in these patients, with a loss in function as well as in quality of life.
The routine preoperative diagnostics include blood examination for C-reactive protein (CRP) and other biomarkers, as well as joint aspirate analysis for cell count, differentiation, and culture. Intraoperative specimens for histology and microbiology are also standard procedure. The microbiological examination of removed implants with sonication, in combination with the implementation of molecular biology techniques in microbiology, represent two novel techniques currently employed to enhance the differential diagnostics of PJI.
We present here the step-wise procedure of analyzing joint aspirate and sonication fluid, using a cartridge-based multiplex polymerase chain reaction (PCR) system. Results were matched against conventional cultures and consensus criteria for PJI. Conventional microbiological cultures from tissue biopsies, joint aspirate and sonication fluid showed a sensitivity of 66.7%, 66.7%, and 88.9%, respectively, and a specificity of 82.3%, 54.6%, and 61.5%, respectively. The PCR diagnostic of the sonication fluid and the joint fluid showed a sensitivity of 50.0% and 55.6%, respectively, and both a specificity of 100.0%. Both PCR diagnostics combined had a sensitivity of 66.7% and a specificity of 100.0%. The multiplex PCR therefore presents a rapid diagnostic tool with moderate sensitivity but high specificity in diagnosing PJI.

Differential diagnostics in painful arthroplasty is crucial for treatment success. Joint aspiration is routinely performed preoperatively. Multiplex polymerase chain reaction of the joint aspirate and the sonication fluid, a feasible tool for quick pathogen detection from these samples is described.

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of ...

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.

Detection of circulating ribonucleic acid (cRNA) from blood is an unmet need in clinical diagnostics. Here we describe methods that characterize cRNA from non-small cell lung cancer patients using sensitive and specific digital polymerase chain reaction. The tests meet design requirements to detect fusion variants within 72 hours.

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Estimates of whole kidney nephron number are important clinically and experimentally, as there is an inverse association between nephron number and an enhanced risk of renal and cardiovascular disease.Herein, the use of the acid maceration method, which provides fast and reliable estimates of whole kidney nephron number, is demonstrated.

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms ...

Ultrasound in Evaluating Diaphragm Dysfunction and Monitoring Treatment

Measuring Diaphragm Thickness and Function Using Point-of-Care Ultrasound

The diaphragm is the main component of the respiratory muscle pump. Diaphragm dysfunction can cause dyspnea and exercise intolerance, and predisposes affected individuals to respiratory failure. In mechanically ventilated patients, the diaphragm is susceptible to atrophy and dysfunction through disuse and other mechanisms. This contributes to failure to wean and poor long-term clinical outcomes. Point-of-care ultrasound provides a valid and reproducible method for evaluating diaphragm thickness and contractile activity (thickening fraction during inspiration) that can be readily employed by clinicians and researchers alike. This article presents best practices for measuring diaphragm thickness and quantifying diaphragm thickening during tidal breathing or maximal inspiration. Once mastered, this technique can be used to diagnose and prognosticate diaphragm dysfunction, and guide and monitor response to treatment over time in both healthy individuals and acute or chronically ill patients.

Author Spotlight: Unraveling the Impact of Mechanical Ventilation on Diaphragm Function and Patient Outcomes 

Diaphragm thickness and function can be assessed in healthy individuals and critically ill patients using point-of-care ultrasound. This technique offers an accurate, reproducible, feasible, and well-tolerated method for evaluating diaphragm structure and function.

The diaphragm is the main component of the respiratory muscle pump. Diaphragm dysfunction can cause dyspnea and exercise intolerance, and predisposes ...

Enhancing Stroke Rehabilitation with Virtual Reality-Based Occupational Training

Cognitive Function and Upper Limb Rehabilitation Training Post-Stroke Using a Digital Occupational Training System

Stroke rehabilitation often requires frequent and intensive therapy to improve functional recovery. Virtual reality (VR) technology has shown the potential to meet these demands by providing engaging and motivating therapy options. The digital occupational training system is a VR application that utilizes cutting-edge technologies, including multi-touch screens, virtual reality, and human-computer interaction, to offer diverse training techniques for advanced cognitive capacity and hand-eye coordination abilities. The objective of this study was to assess the effectiveness of this program in enhancing cognitive function and upper extremity rehabilitation in stroke patients. The training and assessment consist of five cognitive modules covering perception, attention, memory, logical reasoning, and calculation, along with hand-eye coordination training. This research indicates that after eight weeks of training, the digital occupational training system can significantly improve cognitive function, daily living skills, attention, and self-care abilities in stroke patients. This software can be employed as a time-saving and clinically effective rehabilitation aid to complement traditional one-on-one occupational therapy sessions. In summary, the digital occupational training system shows promise and offers potential financial benefits as a tool to support the functional recovery of stroke patients.

Author Spotlight: Rehabilitation of Stroke Patients With a Digital Occupational Training System

The current protocol outlines how the VR-based digital occupational training system enhances the rehabilitation of patients with cognitive impairment and upper limb dysfunction following a stroke.