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 ...

Universidad de Cartagena UDEC

Research

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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

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

RNA干扰在蜱

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

科研

免疫与感染

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

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 ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

实时颤振诱发的粪便中 CWD 朊的转化检测

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

评估介入疗法对人体粪便微生物群影响的体内批处理培养模型

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...