Name: a complete pipeline for isolating and sequencing micrornas, and analyzing them using open source tools
Uploaded: 2019-08-21T13:30:00
Description: Watch this Scientific Journal Video about A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools at JoVE.com

我们要感谢安德鲁·火和马克·凯实验室的成员的指导和建议。

动物实验得到了华盛顿大学机构动物护理和使用委员会的授权。
小型RNA库制备
1. RNA分离
<ol><li>使用标准RNA分离试剂或用于微RNA的试剂盒从生物源分离RNA。对于组织，最好从样品开始，用液氮冷冻样品，然后用预冷冻砂浆和虫子将样品冷冻到粉末中。</li>
	<li>在可量化RNA并提供RNA完整性数（RIN）的仪器上测量每个样品的RNA完整性。...

<ol>
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D., 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=Comprehensive+multi-center+assessment+of+small+RNA-seq+methods+for+quantitative+miRNA+profiling.">Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling.</a> Nature Biotechnology. 36 (8), 746-757 (2018).</li><li>Dard-Dascot, 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=Systematic+comparison+of+small+RNA+library+preparation+protocols+for+next-generation+sequencing.">Systematic comparison of small RNA library preparation protocols for next-generation sequencing.</a> BMC Genomics. 19 (1), 118 (2018).</li><li>Yeri, 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=Evaluation+of+commercially+available+small+RNASeq+library+preparation+kits+using+low+input+RNA.">Evaluation of commercially available small RNASeq library preparation kits using low input RNA.</a> BMC Genomics. 19 (1), 331 (2018).</li><li>Coenen-Stass, A. M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+methodologies+for+microRNA+biomarker+detection+by+next+generation+sequencing.">Evaluation of methodologies for microRNA biomarker detection by next generation sequencing.</a> RNA Biology. 15 (8), 1133-1145 (2018).</li><li>Baran-Gale, 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=Addressing+Bias+in+Small+RNA+Library+Preparation+for+Sequencing:+A+New+Protocol+Recovers+MicroRNAs+that+Evade+Capture+by+Current+Methods.">Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods.</a> Frontiers in Genetics. 6, 352 (2015).</li><li>Jayaprakash, A. D., Jabado, O., Brown, B. D., Sachidanandam, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+remediation+of+biases+in+the+activity+of+RNA+ligases+in+small-RNA+deep+sequencing.">Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing.</a> Nucleic Acids Research. 39 (21), e141 (2011).</li><li>Van Nieuwerburgh, 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=Quantitative+bias+in+Illumina+TruSeq+and+a+novel+post+amplification+barcoding+strategy+for+multiplexed+DNA+and+small+RNA+deep+sequencing.">Quantitative bias in Illumina TruSeq and a novel post amplification barcoding strategy for multiplexed DNA and small RNA deep sequencing.</a> PLoS One. 6 (10), e26969 (2011).</li><li>Valdmanis, P. N., 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=Upregulation+of+the+microRNA+cluster+at+the+Dlk1-Dio3+locus+in+lung+adenocarcinoma.">Upregulation of the microRNA cluster at the Dlk1-Dio3 locus in lung adenocarcinoma.</a> Oncogene. 34 (1), 94-103 (2015).</li><li>Schwarzenbach, H., da Silva, A. M., Calin, G., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+Normalization+Strategies+for+MicroRNA+Quantification.">Data Normalization Strategies for MicroRNA Quantification.</a> Clinical Chemistry. 61 (11), 1333-1342 (2015).</li><li>Viollet, S., Fuchs, R. T., Munafo, D. B., Zhuang, F., Robb, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T4+RNA+ligase+2+truncated+active+site+mutants:+improved+tools+for+RNA+analysis.">T4 RNA ligase 2 truncated active site mutants: improved tools for RNA analysis.</a> BMC Biotechnology. 11, 72 (2011).</li><li>Chiang, H. 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=Mammalian+microRNAs:+experimental+evaluation+of+novel+and+previously+annotated+genes.">Mammalian microRNAs: experimental evaluation of novel and previously annotated genes.</a> Genes &amp; Development. 24 (10), 992-1009 (2010).</li><li>Fromm, 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=A+Uniform+System+for+the+Annotation+of+Vertebrate+microRNA+Genes+and+the+Evolution+of+the+Human+microRNAome.">A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome.</a> Annual Review of Genetics. 49, 213-242 (2015).</li></ol>

尽管20多年前鉴定了微RNA，但微RNA测序过程仍然十分费力，需要专门的设备，阻碍了实验室常规采用内部协议14。其他技术可以同时评估微RNA，如微RNA微阵列和多路复用表达面板;然而，这些方法是有限的，因为它们只量化其探针集中存在的微RNA。因此，他们忽略了小RNA测序的重要特征，如新微RNA的鉴定，以及微RNA等形式-核苷变化，这些改变可以产生重要的生物?...

人类基因组2中首次发现于1993年1，现在估计有近2000个微RNA。微RNA是小型非编码RNA，通常为21-24个核苷酸长。它们是基因表达的转录后调节器，通常结合目标基因的3个未翻译区域（3-UTR）的互补位点，以抑制蛋白质表达和降解mRNA。量化微RNA可以给基因表达提供有价值的见解，并为此开发了几种协议3。
我们开发了一种定义、可重复和长期的协议，用于小型RNA测序，以及使用开源生物信息学工具分析规范化读取。重要的是，我们的协议能够同时识别内源性微RNA和外源交付的构造，从而产生类似微RNA的物种，同时最大限度地减少到其他小RNA物种（包括核糖体RNA）的读数。rRNA），转移RNA衍生的小RNA（tsRNA），重复衍生的小RNA和mRNA降解产物。幸运的是，微RNA是5-磷酸化和2-3羟基化4，一个功能，可以利用它们从这些其他小RNA和mRNA降解产物分离。对于微RNA克隆和测序，存在若干商业选择，这些选择往往更快、更容易进行多路复用;然而，试剂盒的专有特性及其频繁的修改使得比较样品运行具有挑战性。我们的策略通过丙烯酰胺和阿甘蔗凝胶纯化步骤，仅优化收集正确大小的微RNA。在此协议中，我们还描述了使用开源工具将序列读取与微RNA对齐的过程。这套说明对于信息学新手用户特别有用，无论我们使用的是库准备方法还是商业方法。
该协议已用于几个已发表的研究。例如，它被用来识别Dicer酶在距离茎环结构内部回路两个核苷酸的距离上切割小发夹RNA的机制，即所谓的"循环计数规则"5。我们还遵循这些方法，以识别从重组的阿德诺相关病毒载体 （rAAV） 表达的小发夹 RNA （shRNA） 的相对丰度，以识别在肝脏之前可以耐受的 shRNA 表达阈值毒性与过量shRNA表达6相关。利用这个协议，我们还确定了肝脏中的微RNA，这些微RNA对缺乏微RNA-122（一种高度表达的肝微RNA）作出反应，同时也确定了这种微RNA7的降解模式。由于我们在众多实验中一致地使用了我们的协议，因此我们能够纵向观察样品制剂，并且发现没有明显的批次效应。
在共享该协议时，我们的目标是使用户能够使用负担得起的设备和试剂以及免费的生物信息学工具，在几乎任何组织或细胞系中生成高质量、可重复的微RNA定量。

<table>
	<tbody>
		<tr>
			<td>100 bp DNA ladder</td>
			<td>NEB</td>
			<td>N3231</td>
			<td></td>
		</tr>
		<tr>
			<td>19:1 bis-acrylamide</td>
			<td>Millipore Sigma</td>
			<td>A9926</td>
			<td></td>
		</tr>
		<tr>
			<td>25 bp DNA step ladder</td>
			<td>Promega</td>
			<td>G4511</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid phenol/chloroform</td>
			<td>ThermoFisher</td>
			<td>AM9720</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide RNA loading dye</td>
			<td>ThermoFisher</td>
			<td>R0641</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Biorad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer instrument</td>
			<td>Agilent</td>
			<td>G2991AA</td>
			<td>For assessing RNA quality and concentration</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (100%)</td>
			<td>Sigma</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Buffer II</td>
			<td>ThermoFisher</td>
			<td>AM8547</td>
			<td></td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>ThermoFisher</td>
			<td>AM9516</td>
			<td>Blue color helps in visualizing pellet</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma</td>
			<td>P5958</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxi Vertical Gel Box 20 x 20cm</td>
			<td>Genesee</td>
			<td>45-109</td>
			<td></td>
		</tr>
		<tr>
			<td>miRVana microRNA isolation kit</td>
			<td>ThermoFisher</td>
			<td>AM1560</td>
			<td></td>
		</tr>
		<tr>
			<td>miSeq system</td>
			<td>Illumina</td>
			<td>SY-410-1003</td>
			<td>For generating small RNA sequencing data</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nusieve low-melting agarose</td>
			<td>Lonza</td>
			<td>50081</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm (laboratory sealing film)</td>
			<td>Millipore Sigma</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-ethylene glycol 8000</td>
			<td>NEB</td>
			<td>included with M0204</td>
			<td></td>
		</tr>
		<tr>
			<td>ProtoScript II First strand cDNA Synthesis Kit</td>
			<td>NEB</td>
			<td>E6560S</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Fluorometer</td>
			<td>ThermoFisher</td>
			<td>Q33226</td>
			<td>For quantifying DNA concentration</td>
		</tr>
		<tr>
			<td>Qubit RNA HS Assay kit</td>
			<td>ThermoFisher</td>
			<td>Q32855</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Fisher Scientific</td>
			<td>12640</td>
			<td></td>
		</tr>
		<tr>
			<td>Siliconized Low-Retention 1.5 ml tubes</td>
			<td>Fisher Scientific</td>
			<td>02-681-331</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA ligase 1</td>
			<td>NEB</td>
			<td>M0204</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA Ligase 2, truncated</td>
			<td>NEB</td>
			<td>M0242S</td>
			<td></td>
		</tr>
		<tr>
			<td>TapeStation</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td>For assessing RNA quality and concentration</td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>NEB</td>
			<td>M0273X</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Biorad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base pH 7.5</td>
			<td>Sigma</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered EDTA</td>
			<td>Sigma</td>
			<td>T9285</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>ThermoFisher</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Ethidium bromide (10 mg/ml)</td>
			<td>Invitrogen</td>
			<td>15585-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal miRNA cloning linker</td>
			<td>NEB</td>
			<td>S1315S</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>U5378</td>
			<td></td>
		</tr>
	</tbody>
</table>

a complete pipeline for isolating and sequencing micrornas, and analyzing them using open source tools

一半的人类记录被认为是由微RNA调节的。因此，量化微RNA表达可以揭示疾病状态的基本机制，并提供治疗靶点和生物标志物。在这里，我们将详细介绍如何准确量化微RNA。简而言之，该方法描述了分离微RNA、将它们与适合高通量测序的适配器、放大最终产品以及准备样本库。然后，我们解释如何将获取的测序读数与微RNA发夹对齐，并量化、规范化和计算其差分表达。多功能且坚固，这种结合的实验工作流程和生物信息分析使用户能够从组织提取开始，完成微RNA定量。

图书馆准备中涉及的步骤图 图2概述了小RNA提取、测序和对齐的总体原理图。 采集了一只雄性小鼠和一只雌性小鼠的肝脏样本，并冷冻在液氮中。提取总RNA并评估质量和浓度。
小RNA测序产生足够的RNA测序 从两个独立的RNA提取中提取的3μgRNA被用作小RNA测序的起始材料。样?...

Watch this Scientific Journal Video about A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools at JoVE.com

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

在这里，我们描述了一个分步策略，用于隔离小型RNA、丰富微RNA和为高通量测序准备样本。然后，我们介绍如何使用开源工具处理序列读取并将其与微RNA对齐。

用于隔离和测序微RNA并使用开源工具分析微RNA的完整管道

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

我们开发了一种定义、可重复和长期的协议，用于小型RNA测序和使用开源生物信息学工具分析规范化读取。我们战略的主要优点是，它通过凝胶纯化准确收集微RNA，无论微RNA如何收集，生物信息学分析都可以应用。微RNA的高通量测序可以揭示对疾病机制、微RNA处理以及提供小型RNA的基因治疗方法的准确定量。确保在无 RNase 的环境中工作，并在整个过程中将所有RNA产品保持在冰上。可视化凝胶切割步骤将有助于查看者识别下游应用所需的产品的正确尺寸。对于三种原生适配器连接，将 11 微升 RNA 与 1.5 微升 ATP 无 ATP 10X T4RNA 连接酶反应缓冲液、1 微升聚乙烯二醇和 0.5 微升三个原成链接器。在热循环器上加热样品在 95 摄氏度下 30 到 40 秒，然后在冰上冷却 1 分钟。加入一微升T4RNA连结酶2，在室温下孵育反应两小时。当样品孵育时，将15%聚丙烯酰胺凝胶倒入0.8毫米分离的玻璃板中，用塑料铸件插入梳子。当凝胶凝固后，在罐中加入0.5 5X TBE，并大力清洗残留尿素的孔。在375伏特的预运行聚丙烯酰胺凝胶25分钟后，将样品加载，在每个样品之间至少留下一个通道。以不对称模式装载至少两组标记的 20 微升，以跟踪凝胶方向，并在恒定的 375 伏特下运行凝胶。15 分钟后，在运行的其余部分增加至恒定的 425 伏特，并运行凝胶约两个小时。在运行结束时，使用板分离器将凝胶从玻璃板中去除，然后将凝胶放在塑料页保护器上。在500微升蒸馏水中稀释5微升溴化乙酰胺，并将染料添加到顶部浅蓝色标记上方的标记通道上。接下来，使用干净的剃须刀刀片在紫外光下将每个通道的凝胶从上部切割到下部标记，然后将碎片转移到四到四厘米方的实验室密封膜中。水平切割约三个切口和两个切口，以产生 12 个小方块。在密封膜上加入 400 微升 0.3 摩尔氯化钠，并将凝胶片引导到 1.5 毫升硅化管中。在四摄氏度下将样品在四摄氏度下搅拌。第二天早上，将每个上流液的400微升转移到新的管子中。每管加入1毫升100%乙醇和1微升15毫克甘油原共沉淀物。然后在零下80摄氏度的温度下存放管子一小时。对于五个主要连接器连接，首先旋转解冻的样品，然后重新暂停水中的颗粒。接下来，在每个样品中加入0.5微升100微摩尔5个原极链接器、1微升T4RNA连结液缓冲液、10毫摩尔ATP微升和1微升聚乙烯二醇。在90摄氏度下加热反应30秒，然后放在冰上。然后将一个微升的T4RNA连接到每个管中，并在室温下孵育反应两小时。对于逆转录，通过离心收集样品，并在8.25微升无核酸酶自由水中重新预支风干颗粒。从互补的DNA合成试剂盒中加入0.5微升100微摩尔逆转录酶底转和5微升2X逆转录酶反应混合物。在42摄氏度下3分钟后，在每个样品中加入1.5微升10倍逆转录酶，在42摄氏度的热循环器中孵育30分钟。中和后， 用29.5微升水、5微升10X taq缓冲液、1微升核三磷酸盐、25微摩尔前底转微升、25微摩尔反向底土2微升、0.5微升塔克聚合酶和10微升反向转录补充DNA进行PCR反应。然后运行两个 PCR 反应。对于阿加罗斯凝胶纯化，准备一个4%的加糖凝胶与低熔化的阿加罗斯和加载40微升或更多PCR产品到凝胶与加载染料和100基对和25基对大小标记。运行凝胶后，将带子切割到 125 基对带上方，然后根据制造商的说明使用凝胶提取套件。在使用适当的设备量化最终序列库之前，在室温下摇动将凝胶带溶解在缓冲液中。然后使用适当的开源生物信息学工具分析序列数据。在这个具有代表性的PCR反应中，低熔糖凝胶，与获得的链接器链接器产品相比，可以观察到获得的正确克隆产品和不饱和样品。与人类微RNA发夹对齐后，可以观察到每个复制的微RNA读取计数之间的强一致性。共检测到306个微RNA物种，其中对微RNA122的读取量最大。重要的是要记住，切割凝胶在适当的大小，使微RNA的准确恢复。在对微RNA进行测序后，用户可以应用生物信息学分析来描述其感兴趣的组织内微RNA的分布特征。该技术已用于识别微RNA的处理方式，以及某些癌症中哪些微RNA被改变。在处理溴化乙基时和在紫外线下看凝胶时，请务必小心。

Research

Education

JoVE Core

JoVE Journal

Cell Biology

Genetics

Unit 1

Control of Gene Expression

SCIENTISTS IN ACTION

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

科研

遗传学

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

有针对性的下一代测序和生物信息学管道评价宪政疾病的遗传决定因素

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

单缓步动物标本的超低输入基因组测序库制备

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

纳米测序全血 mRNA 测序

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...