Name: fecal (micro) rna isolation
Uploaded: 2020-10-28T15:00:00.000+00:00
Duration: 5 min 35 s
Description: Watch this Scientific Journal Video about Fecal micro RNA Isolation at JoVE.com

我们得到了哈佛医学院生物聚合物设施的生物分析仪技术援助。这项工作得到了国家多发性硬化症协会研究资助RG-1707-28516（H.L.W.和S.L.）的支持。

这里描述的所有涉及研究动物的方法均已获得哈佛医学院布莱根妇女医院机构动物护理和使用委员会（IACUC）的批准。
此处描述的所有涉及人类研究主题的方法均符合合作伙伴人类研究委员会制定的指导方针。
1. 粪便样本采集
<ol><li>高压灭菌或为实验中的每个动物受试者制备带有螺旋盖的无菌且无核酸酶的 2 mL 微量离心管。<ol><li>对于人类受试者，为每个受试者提供适当的、无核酸酶的无菌粪便标本采集装置。</li>
	</ol></li>
	<li>在无菌环境中从每个动物受试者收集25-100mg（小鼠粪便样品约1-4个粪便颗粒）粪便样品。 注意:两个或多个粪便颗粒（~50mg或更重）是获得最高纯度RNA的首选。<ol><li>利用所有必要的个人防护设备 （PPE） 和材料，例如:一副实验室手套、消毒喷雾剂和无菌纸巾，对放置动物研究对象的工作区域进行消毒，以避免粪便样本污染。<ol><li>对于人类受试者，指示每个研究对象/收集者在尽可能无菌的环境中收集100-200毫克粪便样本。使用标准无菌操作，避免粪便样本污染。</li>
		</ol></li>
	</ol></li>
	<li>将每个动物受试者的粪便样本直接收集到带有螺旋盖的 2 mL 微量离心管中，不要接触任何其他表面以避免污染。<ol><li>对于人类受试者，指示每个受试者直接排便到提供的适用收集装置（例如，无菌粪便标本采集套件）中以避免污染。 注意:指示受试者避免被马桶表面、水、尿液或任何其他非无菌表面/物体污染。</li>
	</ol></li>
	<li>立即在-80°C下冷冻收集在2 mL微量离心管中的粪便样品，或在粪便重悬之前放入干冰桶中以获得更好的RNA数量和质量，如下所述。<ol><li>对于人类粪便标本，将每个200mg的新鲜标本等分到带螺旋盖的2mL微量离心管中，并在粪便重悬之前将其冷冻至-80°C，如下所述。<ol><li>对于不在带螺旋盖的 2 mL 微量离心管中的粪便标本的储存，在粪便重悬之前，称量每个冷冻标本 100-200 mg 并将其转移到单独的带螺旋盖的 2 mL 微量离心管中，如下所述。 注意:对于人类粪便标本，请避免服用超过200毫克的粪便标本进行RNA分离，因为它可能会在以下步骤中造成困难。将超载样品放入 2 mL 微量离心管中时，水相、有机相和中间相可能无法清晰形成和分离。该过程可以在此处暂停。</li>
		</ol></li>
	</ol></li>
</ol>2. 洗涤液的制备
<ol><li>将 21 mL 美国化学学会 （ACS） 级 100% 乙醇加入 miRNA 分离试剂盒中提供的洗涤溶液 1（参见 材料表）中，以达到 30 mL 的最终体积，如瓶子上所示。涡旋直到所有东西都溶解在瓶子里。</li>
	<li>将 40 mL ACS 级 100% 乙醇加入提供的洗涤溶液 2/3 中，以达到 50 mL 的最终体积，如瓶子上所示。涡旋5秒或直到最终混合物充分混合。</li>
</ol>3. 设备和材料的准备
<ol><li>用核糖核酸酶（RNase）去污溶液（例如市售RNase去污溶液）喷洒工作区域和设备，例如:实验室工作台、化学通风橱和微量离心管架。为避免污染，请在必要时随时随地将RNase去污溶液涂抹在表面上。</li>
	<li>穿上干净的实验室外套，戴上口罩，戴上适当的实验室手套，以保护粪便样品中的RNA免受人体皮肤上存在的核酸酶的影响。用RNase去污溶液喷洒手套，并经常更换手套以避免污染。</li>
	<li>为储存在-80°C的粪便样品准备一桶干冰，以防止在粪便重悬之前解冻，并为材料准备一桶冰，例如:酸苯酚:氯仿，以延长保质期。 注意:确保材料（包括协议中使用的培养基）是无菌的，没有核酸酶污染。</li>
</ol>4.粪便重悬
<ol><li>将 25-100 mg 粪便样品重悬于 600 μL 无菌 1x Dulbecco 磷酸盐缓冲盐水 （DPBS） 中。 注意:粪便样品从-80°C解冻时应立即处理，甚至不部分解冻，以尽量减少RNA酶和细胞RNA的释放，因为当样品中的细胞解冻时，冰晶会破裂内部和外部细胞区室。<ol><li>在室温 （RT） 下将 600 μL 1x DPBS 加入带有螺旋盖的 2 mL 微量离心管中，其中包含粪便样品。</li>
		<li>将粪便样品混合物浸没在 600 μL 1x DPBS 中，在室温下盖上盖子的 2 mL 微量离心管中孵育 30 分钟。</li>
		<li>用 1 mL 移液器吸头捣碎重悬混合物，并在盖上 2 mL 微量离心管的情况下涡旋良好。为了优化和增加RNA的数量和质量，用均质机重悬混合物，并在S4000（或4000rpm）和45秒下设置一个循环。</li>
	</ol></li>
</ol>5. 有机萃取
注意:使用危险化学品通风柜进行以下步骤，直到步骤6使用酸苯酚:氯仿和ACS级100%乙醇，因为它们具有毒性和易燃性。处理危险材料时，请根据需要更换个人防护装备，并遵循适当的标准预防措施。
<ol><li>用 600 μL 酸性苯酚:氯仿提取 RNA（所需的酸性苯酚:氯仿的体积等于步骤 4.1 中加入的 1x DPBS 的初始体积）。<ol><li>向步骤 4.1 中的悬浮液中加入 600 μL 酸苯酚:氯仿。 注意:从瓶子的下相取出酸苯酚:氯仿从瓶子的下相中取出，因为上相与水性缓冲液混合。如果这两个相之间的中间相受到干扰，则仅当中间相重新建立以避免污染时，才等待并取出酸苯酚:氯仿。</li>
	</ol></li>
	<li>将混合物涡旋60秒以彻底混合。或者，为了优化和增加产量中RNA的数量，使用均质器混合，在S4000和45 s下设置一个循环。</li>
	<li>在室温下以10，000× g 离心15分钟，用微量离心机分离水相和有机相。离心后，界面应紧凑。如果没有，请重复离心。 注意:如果由于初始体积与添加的酸苯酚:氯仿的体积比在多次重复离心后无法达到预期的那样紧凑，请继续回收水相，更加小心以避免污染。</li>
	<li>回收水相并将其转移到带有铰链盖的新 2 mL 微量离心管中（miRNA 分离试剂盒不提供）。<ol><li>小心地取出水相（或上相），不要干扰下相，并将其转移到带有铰链盖的新 2 mL 微量离心管中。注意转移的体积（例如，~500 μL）。 注意:当中间相致密且上层相明显分离时，水相顶部可能漂浮着一些微小的残留颗粒。小心移液以避免这些残留物，并且仅恢复可见且清晰分离的水相，以确保高质量的RNA产量，即使您只能获得少量的水相。</li>
	</ol></li>
</ol>6. 最终RNA分离
<ol><li>向 2 mL 微量离心管中的水相中加入 1.25 体积的 RT ACS 级 100% 乙醇（例如，如果从步骤 5.4 中回收 500 μL 水相，则加入 625 μL 100% 乙醇）。涡旋3秒。</li>
	<li>通过miRNA分离试剂盒中提供的滤芯加载水相/乙醇混合物。<ol><li>对于每个样品，将滤芯放入试剂盒提供的收集管之一中。<ol><li>移液并将 600 μL 水相/乙醇混合物上样到滤芯中。 注意:移液前短暂涡旋混合物，将乙醇与水相彻底混合。一次上样的水相/乙醇混合物不得超过 700 μL。</li>
		</ol></li>
		<li>以 10，000 x g 离心 90 秒以过滤混合物。以更高的速度旋转可能会损坏过滤器。</li>
		<li>弃去滤液并重复步骤6.2.1至6.2.2，直到所有混合物在连续应用中通过相同的滤膜过滤。保留并重复使用相同的收集管进行下面的洗涤步骤。</li>
		<li>用 700 μL miRNA 洗涤溶液 1 洗涤过滤器。 注意:miRNA 洗涤液 1 含有硫氰酸胍，可引起皮肤刺激和严重眼睛损伤。穿戴必要的个人防护用品，例如:手套、面罩、实验室防护服。必要时经常更换手套。<ol><li>将 700 μL miRNA 洗涤溶液 1（用 ACS 级 100% 乙醇制备的工作溶液）涂入滤芯中。</li>
			<li>离心60秒，通过滤芯过滤miRNA洗涤溶液1。</li>
			<li>从收集管中丢弃滤液，并将相同的滤芯放入同一收集管中。</li>
		</ol></li>
		<li>用洗涤溶液 2/3 洗涤过滤器，每次一次，体积分别为 700 μL、500 μL 和 250 μL。<ol><li>将 700 μL 洗涤溶液 2/3（用 ACS 级 100% 乙醇制备的工作溶液）加入滤芯中。<ol><li>以10，000× g 离心1分钟。</li>
				<li>从收集管中丢弃滤液，并将相同的滤芯放入同一收集管中。</li>
			</ol></li>
			<li>将 500 μL 洗涤溶液 2/3 加入滤芯中。<ol><li>以10，000× g 离心1分钟。</li>
				<li>从收集管中丢弃滤液，并将相同的滤芯放入同一收集管中。</li>
			</ol></li>
			<li>将 250 μL 洗涤溶液 2/3 加入滤芯中。<ol><li>以10，000× g 离心1分钟。</li>
				<li>从收集管中丢弃滤液。</li>
			</ol></li>
			<li>将滤芯转移到新的收集管中，旋转组件5分钟以去除过滤器中的残留液体。</li>
		</ol></li>
	</ol></li>
</ol>7. 用 50 μL 无核酸酶水洗脱 RNA
<ol><li>将滤芯转移到新的收集管中。将 50 μL 无核酸酶水移液到过滤器中心并盖上收集管。<ol><li>在室温下孵育10分钟。</li>
		<li>以8，000 x g 旋转5分钟，将RNA回收到新的收集管中。</li>
		<li>使用荧光计测定回收粪便RNA的浓度和纯度。回收的粪便RNA可以储存在-80°C。</li>
	</ol></li>
</ol>

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作者声明与本协议论文中描述的研究方法无关的相关或重大经济利益。

重要的是使用无RNase技术来防止分离过程中的RNase污染7。离心并形成致密界面后，在回收水相时，关键是要避免中间相、下层相和颗粒污染物漂浮在水相顶部。此外，还添加了 500 μL 和 250 μL 洗涤溶液 2/3 的两个洗涤步骤，以消除滤膜中的污染物，从而优化质量。此外，不建议使用超过200 mg的起始样品材料，因为它可能会在中间相的明确形成中造成困难。同样，不建议使用小于25 ...

细胞外RNA被认为是介导许多生物过程的重要因素1。粪便中的细胞外RNA于2008年首次被报道为结肠癌和活动性溃疡性结肠炎2的标志物，最近发现它是肠腔和粪便的正常成分，并介导宿主 -微生物通讯3，4，5。该RNA分离方案的目的是从动物和人类受试者的粪便样品中提取高质量的细胞外RNA。该方案改编自商业miRNA分离试剂盒。采集的 RNA 用于下游分析，如 RNA 测序、RT-PCR 和微阵列。该协议包括几个重要且有用的技巧，以最大限度地提高动物和人类粪便中发现的RNA的数量和质量。开发和优化这种RNA（包括microRNA）分离方法的原因是减少粪便中的微生物RNA，限制研究中的变量并分析肠道中的RNA组成，而不考虑各种混杂因素和污染源。值得注意的是，这种RNA分离最大限度地减少了活细胞和活微生物（细胞RNA）释放的RNA。它侧重于由肠道细胞释放或通过食物摄入获得的细胞外RNA。原则上，该方法不适用于研究微生物转录组的研究。

<table><tbody><tr><td>Acid-Phenol: Chloroform, pH 4.5 (with IAA, 125:24:1)</td><td>Thermo Fischer Scientific</td><td>AM9720</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fischer Scientific</td><td>14190-144</td><td></td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>mirVana miRNA Isolation Kit</td><td>Thermo Fischer Scientific</td><td>AM1561</td><td></td></tr><tr><td>Nuclease-Free microcentrifuge tubes (1.5 mL, 2 mL)</td><td></td><td></td><td></td></tr><tr><td>Nuclease-Free Water (Not DEPC-treated)</td><td>Thermo Fischer Scientific</td><td>AM9937</td><td></td></tr><tr><td>Pipettor and Nuclease-Free Pipette tips (with filter)</td><td></td><td></td><td></td></tr><tr><td>PowerLyzer 24 Homogenizer</td><td>QIAGEN</td><td>13155</td><td></td></tr><tr><td>RNaseZap RNase Decontamination Solution</td><td>Thermo Fischer Scientific</td><td>AM9780</td><td></td></tr><tr><td>Vortex Shaker</td><td></td><td></td><td></td></tr></tbody></table>

fecal (micro) rna isolation

越来越清楚的是，RNA存在于动物和人类的肠腔和粪便中。下面描述的方案从动物和人类受试者的粪便样本中分离出包括microRNA在内的总RNA。目的是分离高纯度和高数量的总RNA，用于下游分析，如RNA测序、RT-PCR和微阵列。这种优化方案在miRNA分离中的优势在于能够通过描述的额外洗涤步骤分离高度纯化的RNA产物，通过改进的样品重悬方法增加获得的RNA数量，以及重要的去污技巧。一个限制是无法处理和纯化超过200 mg的较大样品，因为这些样品量会导致界面的清晰形成困难。因此，大样品量可能会污染方案中描述的待提取水相，有机物会影响最终分离的RNA的质量。然而，从高达 200 mg 的样品中分离出的 RNA 足以用于大多数下游分析。

分别从50 mg小鼠粪便样品（2个小鼠粪便沉淀）和100 mg人粪便标本中分离代表性RNA，并在50 μL无核酸酶水中洗脱。分光光度计对浓度的分析表明，分别分离出49μg和16μgRNA的总量（表1）。如 ~2.0 的 A260/A280 比值和 ~1.8 的 A260/A230 比值所示，RNA 纯度很高（表 1）。如报告3所示，粪便中的大多数RNA是microRNA，这些microRNA可以存在于外泌体中。与此一致，基于芯片?...

Watch this Scientific Journal Video about Fecal micro RNA Isolation at JoVE.com

Fecal micro RNA Isolation

该协议从动物和人类受试者的粪便样本中分离出高质量的总RNA。商业 miRNA 分离试剂盒具有显著适应性，以优化数量和质量分离纯 RNA。RNA分离株适用于大多数下游RNA检测，如测序、微阵列和RT-PCR。

粪便（微量）RNA分离

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs ...

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Immunology and Infection

Unit 1

Fecal (micro) RNA Isolation

Diagnostic and Radiological Studies of the GI System

SCIENTISTS IN ACTION

4.5K 次观看.  Brigham and Women's Hospital and Harvard Medical School. 该协议从动物和人类受试者的粪便样本中分离出高质量的总RNA。商业 miRNA 分离试剂盒具有显著适应性，以优化数量和质量分离纯 RNA。RNA分离株适用于大多数下游RNA检测，如测序、微阵列和RT-PCR。

视频: Fecal micro RNA Isolation

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

科研

遗传学

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

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

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, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

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, ...

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

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

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 ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

医学

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

生物工程

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

分离和剖析人力胆汁含外来体的MicroRNA-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

外来体的纯化和microRNA剖析来源于血液和文化传媒

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

生物学

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

从蜱 离体 唾液腺培养物和细胞外囊泡中分离microRNA

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

粪便（微量）RNA分离

摘要

探索更多视频

此视频中的章节

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

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

脑脊液的microRNA分析采用实时定量PCR技术

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

分离和剖析人力胆汁含外来体的MicroRNA-

外来体的纯化和microRNA剖析来源于血液和文化传媒

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

从蜱离体唾液腺培养物和细胞外囊泡中分离microRNA

粪便（微量）RNA分离

摘要

探索更多视频

此视频中的章节

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

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

脑脊液的microRNA分析采用实时定量PCR技术

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

分离和剖析人力胆汁含外来体的MicroRNA-

外来体的纯化和microRNA剖析来源于血液和文化传媒

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

从蜱 离体 唾液腺培养物和细胞外囊泡中分离microRNA

从蜱离体唾液腺培养物和细胞外囊泡中分离microRNA