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

4.7K 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

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

科研

生物化学

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

遗传学

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

从海岸沉积物和16S量化的rRNA基因和转录同时DNA-RNA提取采用Real-time PCR检测

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

环境科学

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

禽流感监测与FTA卡：实地方法，生物安全，和运输问题解决了

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

免疫与感染

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

采用TaqMan从儿童凳诺如病毒的检测和Genogrouping一步法RT-PCR技术

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

医学

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

生物学

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...