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

感谢L Bozhilova博士对液滴生成PCR数据的统计分析的建议。感谢H Zhang博士提供用于生成 图3C 和 图4B中的数据的卵母细胞。这项工作由剑桥大学医学研究委员会线粒体生物学部门（MC_UU_00015/9）的SPB进行，并由PFC持有的威康信托基金会首席研究奖学金（212219 / Z / 18 / Z）资助。

所有实验均遵循ARRIVE指南，并获得了剑桥大学动物福利伦理审查机构（AWERB）的批准。
注意：液滴生成之前的所有样品制备步骤必须在干净的PCR前工作区进行，最好尽可能在紫外线灭菌的柜子中进行。此处描述的方案使用特定的液滴生成PCR设备（参见 材料表），虽然通用方法应适用于其他系统，但建议参考制造商关于引物/探针浓度，PCR循环条件等的指南，因为?...

<ol>
	<li>Taanman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+genome:+structure,+transcription,+translation+and+replication.">The mitochondrial genome: structure, transcription, translation and replication.</a> Biochimica Biophysica Acta. 1410 (2), 103-123 (1999).</li><li>Wai, T., 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=The+role+of+mitochondrial+DNA+copy+number+in+mammalian+fertility.">The role of mitochondrial DNA copy number in mammalian fertility.</a> Biology of Reproduction. 83 (1), 52-62 (2010).</li><li>D'Erchia, A. M., 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=Tissue-specific+mtDNA+abundance+from+exome+data+and+its+correlation+with+mitochondrial+transcription,+mass+and+respiratory+activity.">Tissue-specific mtDNA abundance from exome data and its correlation with mitochondrial transcription, mass and respiratory activity.</a> Mitochondrion. 20, 13-21 (2015).</li><li>Stewart, J. B., Chinnery, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+mitochondrial+DNA+heteroplasmy:+implications+for+human+health+and+disease.">The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease.</a> Nature Reviews: Genetics. 16 (9), 530-542 (2015).</li><li>Durham, S. E., Samuels, D. C., Cree, L. M., Chinnery, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+levels+of+wild-type+mitochondrial+DNA+maintain+cytochrome+c+oxidase+activity+for+two+pathogenic+mitochondrial+DNA+mutations+but+not+for+m.3243A--&gt;G.">Normal levels of wild-type mitochondrial DNA maintain cytochrome c oxidase activity for two pathogenic mitochondrial DNA mutations but not for m.3243A--&gt;G.</a> American Journal of Human Genetics. 81 (1), 189-195 (2007).</li><li>Liu, H., 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=Wild-type+mitochondrial+DNA+copy+number+in+urinary+cells+as+a+useful+marker+for+diagnosing+severity+of+the+mitochondrial+diseases.">Wild-type mitochondrial DNA copy number in urinary cells as a useful marker for diagnosing severity of the mitochondrial diseases.</a> PloS One. 8 (6), 67146 (2013).</li><li>Filograna, R., 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=Modulation+of+mtDNA+copy+number+ameliorates+the+pathological+consequences+of+a+heteroplasmic+mtDNA+mutation+in+the+mouse.">Modulation of mtDNA copy number ameliorates the pathological consequences of a heteroplasmic mtDNA mutation in the mouse.</a> Science Advances. 5 (4), (2019).</li><li>Wang, Y., 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=The+increase+of+mitochondrial+DNA+content+in+endometrial+adenocarcinoma+cells:+a+quantitative+study+using+laser-captured+microdissected+tissues.">The increase of mitochondrial DNA content in endometrial adenocarcinoma cells: a quantitative study using laser-captured microdissected tissues.</a> Gynecologic Oncology. 98 (1), 104-110 (2005).</li><li>Boulet, L., Karpati, G., Shoubridge, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+threshold+expression+of+the+tRNA(Lys)+mutation+in+skeletal+muscle+of+patients+with+myoclonic+epilepsy+and+ragged-red+fibers+(MERRF).">Distribution and threshold expression of the tRNA(Lys) mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF).</a> American Journal of Human Genetics. 51 (6), 1187-1200 (1992).</li><li>Lee, J., Hyeon, D. Y., Hwang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+multiomics:+technologies+and+data+analysis+methods.">Single-cell multiomics: technologies and data analysis methods.</a> Experimental and Molecular Medicine. 52 (9), 1428-1442 (2020).</li><li>Taylor, S. C., Laperriere, G., Germain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+Digital+PCR+versus+qPCR+for+gene+expression+analysis+with+low+abundant+targets:+from+variable+nonsense+to+publication+quality+data.">Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data.</a> Scientific Reports. 7 (1), 2409 (2017).</li><li>Hindson, C. M., 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=Absolute+quantification+by+droplet+digital+PCR+versus+analog+real-time+PCR.">Absolute quantification by droplet digital PCR versus analog real-time PCR.</a> Nature Methods. 10 (10), 1003-1005 (2013).</li><li>Herbst, A., 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=Digital+PCR+quantitation+of+muscle+mitochondrial+DNA:+age,+fiber+type,+and+mutation-induced+changes.">Digital PCR quantitation of muscle mitochondrial DNA: age, fiber type, and mutation-induced changes.</a> Journals of Gerontology. Series A: Biological Sciences and Medical Sciences. 72 (10), 1327-1333 (2017).</li><li>O'Hara, R., 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=Quantitative+mitochondrial+DNA+copy+number+determination+using+droplet+digital+PCR+with+single-cell+resolution.">Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution.</a> Genome Research. 29 (11), 1878-1888 (2019).</li><li>Diaz, F., 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=Human+mitochondrial+DNA+with+large+deletions+repopulates+organelles+faster+than+full-length+genomes+under+relaxed+copy+number+control.">Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control.</a> Nucleic Acids Research. 30 (21), 4626-4633 (2002).</li><li>Krishnan, K. J., Bender, A., Taylor, R. W., Turnbull, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplex+real-time+PCR+method+to+detect+and+quantify+mitochondrial+DNA+deletions+in+individual+cells.">A multiplex real-time PCR method to detect and quantify mitochondrial DNA deletions in individual cells.</a> Analytical Biochemistry. 370 (1), 127-129 (2007).</li><li>Lowes, H., Pyle, A., Duddy, M., Hudson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+mitochondrial+DNA+in+progressive+multiple+sclerosis.">Cell-free mitochondrial DNA in progressive multiple sclerosis.</a> Mitochondrion. 46, 307-312 (2019).</li><li>Perier, C., 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=Accumulation+of+mitochondrial+DNA+deletions+within+dopaminergic+neurons+triggers+neuroprotective+mechanisms.">Accumulation of mitochondrial DNA deletions within dopaminergic neurons triggers neuroprotective mechanisms.</a> Brain. 136, 2369-2378 (2013).</li><li>Cossarizza, A., 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=Guidelines+for+the+use+of+flow+cytometry+and+cell+sorting+in+immunological+studies+(second+edition).">Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).</a> European Journal of Immunology. 49 (10), 1457 (2019).</li><li>Espina, V., 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=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nature Protocols. 1 (2), 586-603 (2006).</li><li>Cree, L. M., 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=A+reduction+of+mitochondrial+DNA+molecules+during+embryogenesis+explains+the+rapid+segregation+of+genotypes.">A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes.</a> Nature Genetics. 40 (2), 249-254 (2008).</li><li>Belmonte, F. R., 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=Digital+PCR+methods+improve+detection+sensitivity+and+measurement+precision+of+low+abundance+mtDNA+deletions.">Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.</a> Scientific Reports. 6, 25186 (2016).</li><li>Samuels, D. C., Schon, E. A., Chinnery, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+direct+repeats+cause+most+human+mtDNA+deletions.">Two direct repeats cause most human mtDNA deletions.</a> Trends in Genetics. 20 (9), 393-398 (2004).</li><li>Nissanka, N., Minczuk, M., Moraes, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mitochondrial+DNA+deletion+formation.">Mechanisms of mitochondrial DNA deletion formation.</a> Trends in Genetics. 35 (3), 235-244 (2019).</li><li>Macaulay, I. C., 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=Separation+and+parallel+sequencing+of+the+genomes+and+transcriptomes+of+single+cells+using+G&amp;T-seq.">Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&amp;T-seq.</a> Nature Protocols. 11 (11), 2081-2103 (2016).</li><li>Ludwig, L. S., 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=Lineage+tracing+in+humans+enabled+by+mitochondrial+mutations+and+single-cell+genomics.">Lineage tracing in humans enabled by mitochondrial mutations and single-cell genomics.</a> Cell. 176 (6), 1325-1339 (2019).</li><li>Rooney, J. P., 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=PCR+based+determination+of+mitochondrial+DNA+copy+number+in+multiple+species.">PCR based determination of mitochondrial DNA copy number in multiple species.</a> Methods in Molecular Biology. 1241, 23-38 (2015).</li><li>Kamitaki, N., Usher, C. L., McCarroll, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+droplet+digital+PCR+to+analyze+allele-specific+RNA+expression.">Using droplet digital PCR to analyze allele-specific RNA expression.</a> Methods in Molecular Biology. 1768, 401-422 (2018).</li><li>Maeda, R., Kami, D., Maeda, H., Shikuma, A., Gojo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+single+cell+analysis+of+mitochondrial+heteroplasmy+in+mitochondrial+diseases.">High throughput single cell analysis of mitochondrial heteroplasmy in mitochondrial diseases.</a> Scientific Reports. 10 (1), 10821 (2020).</li><li>Quan, P. L., Sauzade, M., Brouzes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dPCR:+A+Technology+Review.">dPCR: A Technology Review.</a> Sensors (Basel). 18 (4), (2018).</li><li>Lin, X., Huang, X., Urmann, K., Xie, X., Hoffmann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+loop-mediated+isothermal+amplification+on+a+commercial+membrane.">Digital loop-mediated isothermal amplification on a commercial membrane.</a> ACS Sensors. 4 (1), 242-249 (2019).</li><li>Li, Z., 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=Fully+integrated+microfluidic+devices+for+qualitative,+quantitative+and+digital+nucleic+acids+testing+at+point+of+care.">Fully integrated microfluidic devices for qualitative, quantitative and digital nucleic acids testing at point of care.</a> Biosensors and Bioelectronics. 177, 112952 (2021).</li></ol>

除上述细胞类型和物种外，此处描述的方案还适用于多种细胞类型和物种，尽管仔细优化新的检测设计将是确保在远离先前验证的引物/探针组合时保持方法准确性和可重复性的关键。在使用单细胞时，确保尽可能准确地进行样品收集（例如，在通过FACS分选细胞时使用严格的单细胞参数）以确保获得的结果真实反映单个细胞的结果，并确保所有准备工作都在干净的PCR前区域进行，以降低发生污染?...

哺乳动物线粒体 （mt）DNA 是一种小的（约 16.5 Kb）环状 DNA 基因组，存在于线粒体基质中，编码 37 个基因，包括两个 rRNA、22 个 tRNA 和 13 个蛋白质编码基因1。与每个细胞包含每个基因的一个（单倍体）或两个（二倍体）拷贝的核基因组不同，mtDNA存在于每个细胞的线粒体中有多个拷贝，范围从数十个拷贝（例如，成熟的精子细胞）到数十万个拷贝（例如，卵母细胞）2，3。这种多拷贝性质的结果是mtDNA基因组中的突变，可能以单核苷酸多态性（SNP），缺失或重复的形式存在，可以在任何给定细胞中以不同的水平存在，占细胞总mtDNA群体的0%至100%。同一细胞中野生型和突变型mtDNA基因组的存在称为异质性，致病性异质性mtDNA突变是线粒体疾病的主要原因，几种常见的神经系统综合征与潜在的异质性mtDNA突变有关4。
导致临床疾病的异质性mtDNA突变可能性的两个关键参数是异质水平和mtDNA拷贝数。许多异质突变表现出阈值效应，生化和临床表型仅在某个异质水平以上变得明显，通常约为80%5，随后随着异质性的进一步增加而恶化4。然而，考虑细胞中存在的mtDNA拷贝数也很重要，因为这将影响在给定异质水平上存在的野生型（即“健康”）mtDNA基因组的数量。对线粒体疾病患者的研究强调了异质性与拷贝数5，6之间相互作用的重要性，Filograna等人最近报道，尽管线粒体疾病小鼠模型中异质性不变，但mtDNA拷贝数增加减轻了症状7。
虽然近年来已经做了很多工作来提高对mtDNA异质性引起的疾病的发病机制和传播的理解，但大部分工作都是在组织而不是细胞水平上进行的，比较平均组织异质水平和拷贝数测量从大量组织活检和血液样本中获得。一些成熟的技术，例如激光捕获显微切割，允许在细胞水平上进行此类测量8，9;然而，最近高通量单细胞分析方法或所谓的“单细胞组学”10的爆炸式增长，对能够在单细胞水平上准确测量这些关键mtDNA参数的方法提出了要求。
液滴生成PCR方法利用微流体技术的最新进展，通过PCR扩增样品DNA中的特定靶标扩增子 来 改进现有定量未知DNA浓度的方法11。与qPCR不同，在qPCR中，样品DNA在单次反应中扩增，PCR产物的相对积累速率作为读数，该方法将初始样品分成数千个单独的液滴，从而将样品DNA分子划分为空间上独立的反应12。随后的PCR反应在每个单独的液滴中进行，PCR产物仅在含有目标DNA的液滴中积累。该反应的结果是液滴池形式的数字输出，这些液滴要么包含目标DNA和扩增的PCR产物的拷贝，要么不包含目标DNA。使用荧光DNA探针或双链（ds）DNA结合染料，可以计数含有扩增产物的液滴，并且“阳性”与“阴性”液滴的比率可用于计算初始样品中存在的DNA拷贝的绝对数量。与从qPCR反应中获得的相对测量相反，这种绝对测量是使该方法可用于准确测量单细胞中mtDNA拷贝数和异质性的关键因素11，最近的几项研究已经为此目的利用液滴生成PCR技术13，14.本文提出了一种测量来自人和小鼠组织的单细胞中mtDNA拷贝数和缺失异质性的方法。

<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

哺乳动物线粒体（mt）DNA是一种小的环状双链线粒体内DNA分子，编码电子传递链的13个亚基。与二倍体核基因组不同，大多数细胞含有更多的mtDNA拷贝，根据细胞类型，从不到100个拷贝到超过20万个拷贝不等。MtDNA拷贝数越来越多地被用作许多与年龄相关的退行性疾病和疾病的生物标志物，因此，mtDNA拷贝数的准确测量正在成为研究和诊断环境中的关键工具。mtDNA中的突变通常以单核苷酸多态性（SNP）或缺失的形式发生，可以存在于细胞内mtDNA的所有拷贝中（称为同质），也可以作为突变和WT mtDNA拷贝的混合物（称为异质）。异质性mtDNA突变是临床线粒体病理学的主要原因，无论是在罕见疾病中还是在越来越多的常见迟发性疾病（如帕金森病）中。确定细胞中存在的异质性水平是诊断罕见线粒体疾病和旨在了解线粒体可能发挥作用的常见迟发性疾病的研究的关键步骤。传统上，MtDNA拷贝数和异质性通过基于定量（q）PCR的测定或深度测序来测量。然而，最近引入的ddPCR技术为测量这两个参数提供了一种替代方法。与现有方法相比，它具有几个优点，包括能够测量绝对mtDNA拷贝数和足够的灵敏度，即使在低拷贝数下也能从单个细胞进行准确测量。这里介绍的是描述使用ddPCR测量单细胞中mtDNA拷贝数的详细方案，此后称为液滴生成PCR，可以选择同时测量具有mtDNA缺失的细胞中的异质性。还讨论了扩展该方法以测量具有mtDNA SNP的细胞中的异质性的可能性。

液滴产生后，可见一层透明的不透明液滴漂浮在每口井的油相顶部（图1B）。在对单细胞进行实验时，输入裂解物中存在去垢剂会对液滴形成产生不利影响。使用2.1.2.中描述的裂解方案，尽管最终样品中存在少量TWEEN-20残留量，但液滴产量仍可常规达到推荐水平10，000以上（图1C）。然而，使用其他裂解缓冲液，例如缓冲液RLT Plus，形成的液滴数量显着减...

Watch this Scientific Journal Video about Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction at JoVE.com

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

在这里，我们提出了一种用于测量单细胞中绝对线粒体（mt）DNA拷贝数和mtDNA缺失异质水平的方案。

使用数字液滴聚合酶链反应测量单细胞线粒体DNA拷贝数和异质性

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

该协议可用于准确测量单个细胞中存在的线粒体DNA拷贝数和某些类型的线粒体DNA突变的水平。以前的方法可以在含有许多细胞的组织样本中准确地进行这些测量。这种技术的主要优点是我们可以在单个细胞中进行相同的测量。这种方法对那些研究线粒体的人特别有用，并将在该领域有广泛的应用。有效分离和裂解单细胞是该技术的关键。以前使用单细胞分选的经验将非常有帮助。首先，如文中所述，在冰上制备不含样品DNA的PCR预混液。短暂涡旋后，将所需体积的制备预混液分配到液滴生成PCR 96孔板的每个孔中，将样品排列在全柱中。接下来，将预混液添加到不完整列中的任何空孔中，以将其用作非模板对照。然后将输入的DNA添加到每个孔中，使每个孔的总体积为22微升。用粘性板密封板，并将其放在2000 RPM的摇床上一分钟。然后将板以 1000 倍 G 在 4 摄氏度下离心一分钟，然后将其放在冰上。对于液滴生成，首先确保液滴发生器已通电，并检查一瓶液滴发生油是否装载到仪器甲板的位置 E。然后单击触摸屏上的配置样品板。选择样品板上包含样品或空白的所有色谱柱后，单击“确定”以确认板配置。接下来，将液滴发生器盒装载到仪器甲板上的位置B，使所有灯都变成绿色，并将一个空的吸头废液槽放入位置C.然后将带盖的过滤器吸头盒装载到仪器台上的位置D，以便所有灯都变成绿色。接下来，在将样品板装载到仪器甲板的位置 F 之前取下粘性密封，使指示灯变为绿色。还将空液滴收集的96孔板装入凉爽的块中，在零下20摄氏度下预冷，在位置G处使灯变绿。单击仪器触摸屏上的开始液滴生成，液滴发生器盖将自动关闭。液滴生成完成后，目视检查样品收集板，以确认每个孔的油相上方是否存在液滴乳液层。接下来，打开板封口机的电源，将温度设置为180摄氏度，密封时间为五秒。让机器达到工作温度。然后将样品收集板放在板封口机的板架上，并在板顶部放置一个新的箔密封，红线朝上。当印版封口机达到其工作温度时，在仪器触摸屏上按弹出。将板架放入板封口机的抽屉后，按封盖关闭抽屉。密封完成后，抽屉将自动打开。取出密封板并将其放在冷块上。然后关闭板封口机。为了运行PCR，将密封的液滴收集板放入具有96深孔块的PCR机器中。PCR完成后，打开液滴读取器和连接的计算机的电源。将装有PCR后样品的板装入液滴读数器板支架中。将铝箔盖放在样品板上的同时，将盖子放在支架的顶部，并用黑色锁定夹将其固定到位。接下来，要打开液滴阅读器盖，请按下打开/关闭按钮。然后将带有样品板的液滴读取板支架装入读取器室，并将其牢固地放在磁性底座上。再次按下打开/关闭按钮以合上盖子。打开分析软件界面。选择“添加板”选项卡，单击“添加板”，然后单击“配置板”。在板信息选项卡中，输入板名称，从超级混合下拉菜单中选择探针的超级混合、无DUTP，然后在保存数据文件下输入文件名 As.In“选井”选项卡，突出显示要分析的孔，然后单击包括所选孔。然后在“井信息”选项卡中，选择要注释的井。从实验类型下拉菜单中选择直接定量。然后填充样品描述、样品类型和目标名称框，并根据需要添加孔注释和/或板注释。然后单击应用。板配置完成后，单击开始运行。对于结果分析，请选择“数据分析”选项卡，然后从“我的数据文件”菜单中打开要分析的文件。然后单击“1D 振幅”选项卡（用于单色实验）或“2D 振幅”选项卡（用于双色实验）。接下来，选择需要阈值的孔。使用阈值线模式工具在幅度图上正负群体之间的间隙中手动应用阈值，确保十字准线的位置使四个液滴群体清晰分开。对所有样本应用阈值后，通过单击“数据表”选项卡的“导入/导出”，然后选择“将可见数据导出为 CSV”，将结果导出为 csv 文件以供进一步分析。输入合适的文件名，然后单击保存。该协议中使用的PCR和液滴读取程序表明，在fam和hex通道中都存在明确分离的阳性和阴性液滴群体。两种探针均呈阳性的线粒体DNA群体也可以与单细胞裂解物区分开来。该方法还表明，对于线粒体DNA缺失的样品，存在双阳性液滴和仅对DNA未缺失部分呈阳性的液滴的混合群体。该方法已成功用于计数多种哺乳动物细胞（包括小鼠卵母细胞和原代小鼠胚胎细胞）中每个细胞的线粒体DNA拷贝数。此外，在具有高缺失异质性的细胞中，发现单探针阳性群体明显大于双阳性群体。然而，在具有低缺失异质性的细胞中，两个群体的大小相似。因此，准确的结果始终确保液滴生成成功，并且使用 1D 或 2D 振幅视图适当地应用阈值。未使用的裂解物可用于其他单细胞测定，例如焦磷酸测序，它可以测量单核苷酸多态性异质性。这项技术的发展使我们能够准确测量线粒体DNA拷贝数和单个细胞中的缺失异质性，这在以前是不可能的。

Research

JoVE Journal

Medicine

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

测量升主动脉刚度<em&gt;在体内</em&gt;小鼠使用超声

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

科研

医学

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

评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

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

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

磷31磁共振波谱分析：一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

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

翻修术中的新诊断: 种植超声和多重聚合酶链反应

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

用数字聚合酶链反应检测循环核糖核酸 ROS1 和 RET 融合记录的血液检测方法

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

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

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

酸化浸渍法评价全肾肾素数

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

通过焦磷酸测序对线粒体基因组中的单核苷酸多态性进行基因分型

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

Unraveling the Impact of Mechanical Ventilation on Diaphragm Function and Patient Outcomes (Video) | JoVE

使用床旁超声测量隔膜厚度和功能

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