Name: quantification of circular rnas using digital droplet pcr
Uploaded: 2022-09-16T14:50:00
Description: Watch this Scientific Journal Video about Quantification of Circular RNAs Using Digital Droplet PCR at JoVE.com

这项工作得到了生命科学研究所的校内资助，DBT研究资助（BT / PR27622 / MED/30 / 1961 / 2018）和授予Amaresh C. Panda的威康信托基金会/ DBT印度联盟奖学金[IA / I / 18 / 2 / 504017]的支持。我们感谢其他实验室成员校对本文。

RNA对核糖核酸酶敏感;因此，所有试剂、仪器和工作空间都应不含RNase，并应小心处理。
1. circRNA的不同引物设计（见 图1）
<ol><li>使用 BEDTools 从 circRNA 注释数据中检索成熟序列，或通过连接背接连接坐标18，19 之间存在的外显子/内含子序列从 UCSC 基因组浏览器中检索成熟序列。</li>
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<ol>
	<li>Salzman, J., Gawad, C., Wang, P. L., Lacayo, N., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNAs+are+the+predominant+transcript+isoform+from+hundreds+of+human+genes+in+diverse+cell+types.">Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types.</a> PLoS One. 7 (2), 30733 (2012).</li><li>Chen, L. L., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+circRNA+biogenesis.">Regulation of circRNA biogenesis.</a> RNA Biology. 12 (4), 381-388 (2015).</li><li>Kristensen, 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=The+biogenesis,+biology+and+characterization+of+circular+RNAs.">The biogenesis, biology and characterization of circular RNAs.</a> Nature Reviews Genetics. 20 (11), 675-691 (2019).</li><li>Chen, X., Zhou, M., Yant, L., Huang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNA+in+disease:+Basic+properties+and+biomedical+relevance.">Circular RNA in disease: Basic properties and biomedical relevance.</a> Wiley Interdisciplinary Reviews. 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B., 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=Evaluation+of+a+droplet+digital+polymerase+chain+reaction+format+for+DNA+copy+number+quantification.">Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification.</a> Analytical Chemistry. 84 (2), 1003-1011 (2012).</li><li>Hayden, R. 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=Comparison+of+droplet+digital+PCR+to+real-time+PCR+for+quantitative+detection+of+cytomegalovirus.">Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus.</a> Journal of Clinical Microbiology. 51 (2), 540-546 (2013).</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, 2409 (2017).</li><li>Hindson, B. J., 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=High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number.">High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.</a> Analytical Chemistry. 83 (22), 8604-8610 (2011).</li><li>Ishak, A., AlRawashdeh, M. M., Esagian, S. M., Nikas, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic,+prognostic,+and+therapeutic+value+of+droplet+digital+PCR+(ddPCR)+in+COVID-19+patients:+A+systematic+review.">Diagnostic, prognostic, and therapeutic value of droplet digital PCR (ddPCR) in COVID-19 patients: A systematic review.</a> Journal of Clinical Medicine. 10 (23), 5712 (2021).</li><li>Li, 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=Application+of+droplet+digital+PCR+to+detect+the+pathogens+of+infectious+diseases.">Application of droplet digital PCR to detect the pathogens of infectious diseases.</a> Bioscience Reports. 38 (6), (2018).</li><li>Lee, B. 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+UCSC+genome+browser+database:+2022+update.">The UCSC genome browser database: 2022 update.</a> Nucleic Acids Research. 50, 1115-1122 (2022).</li><li>Quinlan, A. R., Hall, I. 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在过去的十年中，随着高通量测序技术的发现，CircRNA研究得到了发展。因此，它被认为是未来RNA疗法的潜在分子。此外，已知它可以作为多种疾病的生物标志物，包括癌症和心血管疾病4，8。然而，circRNA的鉴定是棘手的，因为它的丰度低，并且只有一个特定的背剪接连接序列，将其与亲本mRNA9区分开来。自发现以来?...

RNA测序技术和新颖计算算法的最新进展发现了不断增长的非编码RNA家族的新成员，称为环状RNA（circRNA）1。顾名思义，circRNA是一类没有自由末端的单链RNA分子。它们由称为反向剪接的非规范头到尾剪接形成，其中上游剪接受体位点与下游剪接供体位点共价连接，形成稳定的RNA环1，2。这个过程可以由几个因素介导，包括存在于环状外显子上游和下游的倒置Alu重复元件，或者可以通过一些剪接因子或RNA结合蛋白（RBP）介导2，3。仅由外显子或内含子序列产生的环状RNA被归类为外显子circRNA和环状内含子RNA或ci-RNA，而一些外显子circRNA保留内含子并称为外显子-内含子circRNA（EIcircRNA）3，4。circRNA的功能是多方面的，包括海绵miRNA和/或RBP，调节转录以及通过翻译成肽3，5，6，7来调节细胞功能。一些报告强调了circRNA在各种疾病和生理过程中的重要性8。此外，组织特异性表达模式和对核酸外切酶消化的抗性使其成为疾病诊断的功能生物标志物，也可以用作合适的治疗靶点8。考虑到其在调节健康和疾病方面的重要性，准确定量circRNA表达是当务之急。
已经开发了几种生化方法来量化生物样品中的circRNA9。circRNA定量最方便和最广泛接受的方法之一是逆转录，然后使用不同的引物对进行定量聚合酶链反应（RT-qPCR）10。然而，与线性mRNA相比，大多数circRNA的丰度较低，这使得量化它们具有挑战性11。为了克服这个问题，我们试图使用数字微滴PCR（dd-PCR）来准确量化给定样品中circRNA的数量。dd-PCR是一种遵循微流体原理的先进PCR技术;它在油中产生多个水滴，并且PCR作为单独的反应在每个液滴中发生12。反应发生在单个液滴中，并使用液滴读数器进行分析，该读数器给出了目标基因的正或负液滴数12。这是准确定量目的基因的最灵敏技术，即使给定样本中只有一个拷贝。对抑制剂的敏感性降低，精度更高，并且省略了用于定量的参考基因，使其比传统的qPCR13，14，15更具优势。它已被广泛用作对感兴趣的基因进行绝对定量的研究和诊断工具16，17。在这里，我们描述了使用不同引物分化小鼠C2C12肌管和增殖小鼠C2C12成肌细胞中circRNA定量的详细dd-PCR协议。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >1.5 ml microcentifuge tube</td>
			<td >Tarson</td>
			<td >500010</td>
			<td ></td>
		</tr>
		<tr >
			<td >0.2 ml tube strips with cap</td>
			<td >Tarson</td>
			<td >610020, 510073</td>
			<td></td>
		</tr>
		<tr >
			<td >Filter Tips</td>
			<td >Tarson</td>
			<td >528104</td>
			<td></td>
		</tr>
		<tr >
			<td >DNase/RNase-Free Distilled Water</td>
			<td >Thermo Fisher Scientific</td>
			<td >10977023</td>
			<td></td>
		</tr>
		<tr >
			<td >Phosphate-buffered saline (PBS)</td>
			<td >Sigma</td>
			<td >P4417</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell scrapper</td>
			<td >HiMedia</td>
			<td >TCP223</td>
			<td></td>
		</tr>
		<tr >
			<td >Chloroform</td>
			<td >SRL</td>
			<td >96764</td>
			<td></td>
		</tr>
		<tr >
			<td >DNA diluent</td>
			<td >HiMedia</td>
			<td >MB228</td>
			<td></td>
		</tr>
		<tr >
			<td >Random primers</td>
			<td >Thermo Fisher Scientific</td>
			<td >48190011</td>
			<td></td>
		</tr>
		<tr >
			<td >dNTP set</td>
			<td >Thermo Fisher Scientific</td>
			<td >R0181</td>
			<td></td>
		</tr>
		<tr >
			<td >Murine RNase inhibitor</td>
			<td >NEB</td>
			<td >M0314S</td>
			<td></td>
		</tr>
		<tr >
			<td >Maxima reverse transcriptase</td>
			<td >Thermo Fisher Scientific</td>
			<td >EP0743</td>
			<td></td>
		</tr>
		<tr >
			<td >QX200 dd-PCR Evagreen Supermix</td>
			<td >Bio-Rad</td>
			<td >1864033</td>
			<td></td>
		</tr>
		<tr >
			<td >Droplet generation oil for Evagreen</td>
			<td >Bio-Rad</td>
			<td >1864006</td>
			<td></td>
		</tr>
		<tr >
			<td >PCR Plate Heat Seal, foil, pierceable</td>
			<td >Bio-Rad</td>
			<td >1814040</td>
			<td></td>
		</tr>
		<tr >
			<td >DG8 Cartridges and Gaskets</td>
			<td >Bio-Rad</td>
			<td >1864007</td>
			<td></td>
		</tr>
		<tr >
			<td >DG8 Cartridge holder</td>
			<td >Bio-Rad</td>
			<td >1863051</td>
			<td></td>
		</tr>
		<tr >
			<td >QX200 Droplet Generator</td>
			<td >Bio-Rad</td>
			<td >1864002</td>
			<td></td>
		</tr>
		<tr >
			<td >ddPCR 96-Well Plates</td>
			<td >Bio-Rad</td>
			<td >12001925</td>
			<td></td>
		</tr>
		<tr >
			<td >PX1 PCR Plate Sealer</td>
			<td >Bio-Rad</td>
			<td >1814000</td>
			<td></td>
		</tr>
		<tr >
			<td >C1000 Touch Thermal Cycler with 96&#8211;Deep Well Reaction Module</td>
			<td >Bio-Rad</td>
			<td >1851197</td>
			<td></td>
		</tr>
		<tr >
			<td >QX200 Droplet Reader</td>
			<td >Bio-Rad</td>
			<td >1864003</td>
			<td></td>
		</tr>
		<tr >
			<td >Quantasoft Software</td>
			<td >Bio-Rad</td>
			<td >1864011</td>
			<td></td>
		</tr>
		<tr >
			<td >Silica column</td>
			<td >Umbrella Life Science</td>
			<td >38220090</td>
			<td></td>
		</tr>
		<tr >
			<td >UCSC Genome Browser</td>
			<td ></td>
			<td >https://genome.ucsc.edu/</td>
			<td></td>
		</tr>
		<tr >
			<td >Primer 3</td>
			<td ></td>
			<td >https://primer3.ut.ee/</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of circular rnas using digital droplet pcr

数字液滴聚合酶链反应（dd-PCR）是最灵敏的定量方法之一;它将反应分馏成近 20，000 个油包水液滴，PCR 发生在单个液滴中。与传统的实时qPCR相比，dd-PCR具有多个优势，包括检测低丰度靶标的准确性更高，省略了定量的参考基因，消除了样品的技术重复，以及对样品中的抑制剂表现出高弹性。最近，dd-PCR已成为准确定量靶DNA或RNA以进行基因表达分析和诊断的最流行的方法之一。环状RNA（circRNA）是最近发现的共价闭合RNA分子的大家族，缺乏5'和3'末端。它们已被证明通过充当RNA结合蛋白和microRNA的海绵来调节基因表达。此外，circRNA分泌到体液中，它们对核酸外切酶的抗性使它们成为疾病诊断的生物标志物。本文旨在展示如何进行不同的引物设计、RNA 提取、cDNA 合成和 dd-PCR 分析，以准确定量细胞中的特定环状 RNA （circRNA） 水平。总之，我们证明了使用dd-PCR对circRNA的精确定量。

每个样品中circRNA的绝对数量可以从导出的dd-PCR数据中得出。实时定量PCR分析表明 circBnc2 在分化的C2C12肌管中表达差异化（数据未显示）。在这里，我们想检查c2C12成肌细胞和肌管增殖中 circBnc2 的绝对拷贝数。由于 circBnc2 的表达是在两种条件下进行比较的，因此使用相同的试剂和程序同时处理所有用于RNA分离，cDNA合成和PCR的样品非常重要。为了使用dd-PCR反应对circRNA进行绝对定...

Watch this Scientific Journal Video about Quantification of Circular RNAs Using Digital Droplet PCR at JoVE.com

Quantification of Circular RNAs Using Digital Droplet PCR

该协议描述了数字液滴PCR（dd-PCR）的详细方法，用于使用不同的引物精确定量细胞中的环状RNA（circRNA）水平。

使用数字微滴PCR定量环状RNA

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

dd-PCR是一种高度灵敏且准确的靶DNA或RNA绝对定量方法。它已被用于其他常规方法无法给出准确结果的疾病诊断。dd-PCR是检测极低丰度核酸分子的最灵敏技术。与定量PCR不同，dd-PCR不需要任何管家基因控制即可进行定量。由于其灵敏度和准确性，dd-PCR可用作分子诊断和研究的工具，涉及低丰度RNA分子，如microRNA和环状RNA。在使用dd-PCR之前，应进行RT-PCR或RT-qPCR，以确认引物正在扩增其靶基因，而无需任何非特异性PCR扩增。首先，通过将总环状RNA序列长度的最后100个核苷酸连接到环状RNA序列的前100个核苷酸来制备长度为200个核苷酸的PCR模板序列。使用由入门 3 Web 工具设计的引物模板序列。取一个含有约500万个增殖C2C12肌母细胞的10厘米培养皿和一个为期四天的分化C2C12肌管进行RNA分离。用10毫升1X PBS洗涤细胞三次，然后用移液管丢弃PBS。加入一毫升RNA分离试剂，通过剧烈移液裂解细胞。然后，加入200微升氯仿并涡旋15秒。将试管在4摄氏度下以12， 000 G离心10分钟.取400微升上层水层，将其装入硅胶柱上，并在室温下以12， 000 G离心一分钟。将流量通过新管并加入 600 微升 100% 乙醇。加入 20 微升磁性硅胶珠，将管子放在温度混合器上，设置为 25 摄氏度和 1， 200 rpm，持续五分钟。然后，将管子放在磁性支架上 30 秒或直到溶液变清。将磁性硅胶珠重悬于含有90%乙醇的500微升洗涤缓冲液中。将试管放在磁性支架上30秒后，让珠子沉向磁铁并使用移液器丢弃缓冲液。在热混合器上以 50 摄氏度风干管子三分钟，保持盖子打开。加入20微升无核酸酶的水，重悬磁珠。将溶解的RNA收集在新管中，使用分光光度计进行数量和质量评估。使用分光光度计测量RNA浓度，并取一微克RNA进行cDNA合成。将一微克总RNA与1微升dNTP混合物，四微升5X逆转录酶缓冲液，两微升10X逆转录酶随机引物，0.25微升逆转录酶和0.5微升RNase抑制剂混合。使用不含核酸酶的水将体积补足至20微升。将试管置于 25 摄氏度下 10 分钟，然后放置 50 摄氏度一小时。然后将试管置于 85 摄氏度下五分钟以使酶失活。在0.2毫升PCR管或试管中设置22微升的PCR反应，以及阴性对照管和非模板对照管。加入11微升dd-PCR预混液、5.5微升1微摩尔浓度的环状RNA特异性正向和反向引物混合物以及5.5微升无核酸酶水以设置NTC反应管。同样，要设置测试反应管，请加入11微升dd-PCR预混液，5.5微升1微摩尔浓度的环状RNA特异性正向和反向引物混合物以及5.5微升cDNA。为了产生液滴，将20微升PCR混合物从0.2毫升PCR管转移到液滴发生器盒的样品孔中。使用移液管小心地将70微升液滴发生油添加到液滴发生器盒的油井中。用橡胶垫圈盖住液滴发生器滤芯，并将其放入液滴发生器机中，以获得在液滴发生器滤芯的液滴孔中产生的样品油滴混合物。对于PCR扩增，使用移液管将40微升样品油滴混合物从液滴发生器盒的液滴孔转移到96孔PCR板中。用铝箔封口机密封96孔PCR板。将其放入封板机块中，在180摄氏度下预热，然后单击密封以密封PCR板。然后，将板放入dd-PCR热循环仪中，并将盖子温度设置为105摄氏度，升温速率设置为每秒2摄氏度。PCR结束后，将板放在dd-PCR液滴计数器机上，将板架置于正确的位置以计数液滴。打开液滴分析软件，通过输入样品信息来设置运行。点击分析按钮，分析液滴读数完成后的数据。然后，单击 1D 振幅按钮以查看正极和负极液滴。要将正液滴与负液滴分开，请为具有相同目标的所有样品设置一条通用阈值线。单击导出按钮将环状RNA计数数据导出为csv文件。导出的数据显示了每个样品中存在的环状RNA的数量。通过考虑每个反应中使用的cDNA总量，手动计算每个样品中每纳克RNA的环状RNA数量。此处显示了使用QuantaSoft软件的数据分析。除MT1外，所有样品均显示超过12， 000个液滴计数。由于MT1中的总液滴数较低，因此最终数据分析不考虑该样品。有趣的是，与肌母细胞相比，在4天分化肌管条件下，Circ B和C2的表达模式存在明显差异。对Circ B和C2丰度的拷贝数分析表明，3个重复的C2C12成肌细胞每纳克RNA分别为76.8、67和46拷贝，而2个肌管样品的拷贝数分别为每纳克RNA558和610拷贝。条形图中的数据表示两到三个生物学重复的平均值正负标准偏差。ddPCR是测量靶环RNA准确差异表达的最先进方法之一。这种方法将成为未来一年加速环状RNA研究和诊断行业的重要工具。

Research

JoVE Journal

Biology

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

科研

生物学

Rapid PCR Thermocycling using Microscale Thermal Convection

快速PCR热循环利用微型热对流

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

Isolation of Small Noncoding RNAs from Human Serum

小非编码RNA从人血清中分离

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

利用数字图像相关表征的血管组织标本本地菌株

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

从真核细胞染色体外的环状DNA基因组范围内的净化

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

定量实时反转录 PCR 技术对猕猴猴血浆 MicroRNA 水平的绝对量化

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

利用成熟的细胞隔离试剂盒快速分离脂滴器

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

用于SARS-CoV-2检测和定量的两步逆转录液滴数字PCR方案

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...