资助这项研究是由美国国立卫生赠款R01HL105826 K02HL114749（博士Sadayappan），美国心脏协会中西部奖学金; 11PRE7240022（先生Barefield）和13POST14720024（Govindan博士）。作者非常感谢其出色的技术和文献支持在开发检测的协助下，吉尔Clampit的吉米·佩奇博士和约翰·哈德森博士，MSD。

1。 Uniplex cMyBP C含量<ol><li>前一天实验，大衣96以及MSD裸的标准板捕获抗体。对于-C cMyBP，使用的浓度为5微克/毫升稀释于磷酸盐缓冲盐水（PBS）30μl的体积的小鼠单克隆抗cMyBP-C抗体（明胶免费）。 </li><li>由于是疏水性的，吸液管的解决方案以及入下角，然后点击两侧的板块，传播的解决方案，在整个井。 </li><li>板封口板覆盖着，在4°C孵育O / N无晃动。 </li><li>卸下捕获抗体溶液，攻解掉在水槽上方，然后在一叠纸巾。板的非特异性结合是通过加入150微升的5％（重量/体积）的阻断剂（高档BSA）的PBS溶液，以每孔阻塞。 </li><li>密封板，在室温下孵育1小时，，一阵晃动在700转。 </li><li>在阻塞的步骤，准备的标准和amples。标准系列的起始浓度为2000纳克/毫升，以1％（W / V）阻滞剂A / PBS稀释重组cMyBP-C蛋白片段（氨基酸1 - 271）。然后连续阻滞剂在1％（W / V）的A / PBS稀释5倍。共有7标准+ 1空白（1％（W / V）阻滞剂A / PBS单独）使用。 </li><li>取出封闭溶液，并，洗板3倍与150微升的0.05％（体积/体积）Tween-20/PBS。每一次，取出洗净液反相板水槽上方。第三次洗涤步骤后，大力滑动板在水槽上方，并大力拍打板一层纸巾，直到完全干燥。这是关键的一步，孵化量小，任何剩余洗涤液，将大大稀释下孵化。 </li><li>吸取25微升样品和标准入井。密封板，并在RT下孵育，同时以700rpm振摇1小时。 </li><li>在1％阻滞剂A / PBS，准备检测抗体溶液在1微克/毫升。铜STOM cMyBP-C抗体（表位氨基酸2 - 14），与MSD SULFO-TAG标签作为检测抗体。套件可SULFO-TAG标签，使其成为一个相对简单的程序来标记任何抗体。 </li><li>重复洗涤步骤1.4中所描述的步骤。 </li><li>的检测抗体溶液，以每孔中加入25μl，密封板，并在室温下孵育1小时，在板振动筛设置在700rpm。 </li><li>在潜伏期，运行MSD的演示板（板带LED灯），本部门的成像和软件的（参见试剂节），以确保适当的功能。此外，稀释的读缓冲区，这是由MSD，通过添加4x读缓冲区的5毫升〜15毫升的蒸馏水。 </li><li>重复洗涤步骤1.4中所描述的步骤。 </li><li>通过使用多道移液器向每孔中加入150μl的读缓冲。一定要避免气泡（反向移液是一种合适的方法）。除了读缓冲区后，立即扫描板部门成像仪。 </li></ol> 2。 3复杂-C cMyBP，CK-MB和cTnI检测<ol><li> 96孔3复杂的板和稀释剂的解决方案允许在使用前平衡至室温。该板块是预涂为cMyBP-C，CK-MB和cTnI由MSD捕获抗体。 </li><li> 1％（W / V）阻滞剂A / PBS，每孔加入25微升。点选在板的两侧，以确保通过溶液覆盖整个井。 </li><li>用封口膜密封反应板，将板在板振动筛，并以700rpm的速度摇动30分钟。 </li><li>在此期间，从个人校准器系列稀释的制备。因为每个孔有三个捕获抗体三个校准器需要混合串联在使用前稀释。重组cMyBP-C，CK-MB（MSD）和cTnI（MSD）的1％（W / V）阻滞剂A / PBS稀释创建一个样品，2000纳克/毫升100毫微克/毫升-C cMyBP，CK- MB和25 ng / ml的肌钙蛋白I（样品A）。 </li><li>准备至少100微升的最终体积为每个标准。至完成标准系列，连续稀释的样品5倍。使用25微升的样品A在100微升1％（W / V）阻滞剂A / PBS创建示例B.然后用100微升1％（W / V）阻滞剂A / PBS创建样品C的25微升样品B ，等等。人力从16个科目与心肌梗死和16名对照者的血清样本被用来衡量，CK-MB cMyBP-C和cTnI水平。对这些样品进行1％（重量/体积）的阻断剂A / PBS稀释2倍。 </li><li>加入25微升的标准和已经存在于复杂的板的孔中25微升稀释的样品。重要的是，还包括一个空白（稀释剂）。要建立技术，样品装入在技术一式三份。否则，重复的就足够了。 </li><li>再次密封反应板（板盖可重复使用）和孵育的一盘摇床（每分钟700转），在室温下2小时。 </li><li>就在温育结束时，检测抗体溶液的制备。使用的稀释剂是甲在PBS中的1％（重量/体积）的阻断剂。所有THRee值磺酸基标记的检测抗体稀释到终浓度为1μg/ ml的每个。通常在50倍的股票浓度检测抗体。 </li><li>对于一个完整的96 - 孔板中，2700微升的总稀释后的溶液（包括移液误差）。各检测抗体加入54微升​​至2,538微升1％（W / V）阻滞剂A / PBS。 </li><li> 2小时后，删除解决方案，通过大力弹板水槽上方。用150微升的0.05％Tween-20的PBS洗井3次。中加入25μl的检测抗体溶液，每孔用一个多通道的移液管，并在板的两侧，以确保通过溶液覆盖整个井被窃听。 </li><li>密封板和孵化1小时，而震动700转。 </li><li>在潜伏期，运行MSD的演示板（板带LED灯），以确保适当的功能，热像仪和软件部门。还能淡化读缓冲区，这是由MSD，加入5ml 4x读缓冲到15毫升蒸馏水。 </li><li>重复洗涤步骤，在步骤2.8中所述。 </li><li>加入150μl读缓冲区每口井使用多道移液器。避免气泡是至关重要的（反向移液是一种合适的方法）。除了读缓冲区后，立即扫描扇区成像板。 </li></ol>

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正在等待一个完整的专利申请（申请序列号13/464，466，出版号:美国2012/0282618 A1和日期:05/04/12），以确定相关的风险因素与cMyBP-C的降解和释放到人体体液并定量在人体体液中的水平的cMyBP C。

在这项研究中，我们表明多个心脏生物标记物的检测患者血清中的多重免疫测定法的适用性。这种技术有许多优点，比传统的ELISA。首先，使用ECL检测试剂盒中​​的，而不是比色检测。在ECL，电信号刺激当地生产的测量属性（光），从而解偶联信号的刺激事件，从而降低背景14。这允许高灵敏度，可以看出在表2中。 复用不会导致检测的敏感度的降低，?...

低量的蛋白质，在复杂生物样品，如血清，测量病人的管理，以及基础科​​学发展的临床重要性。比如，增加血清心脏生物标志物，如肌钙蛋白I，在临床的设置是一致的急性心肌梗死（MI）1。为了检测血清样品中的蛋白质，标准的酶联免疫吸附试验（ELISA）法是最常用的技术，因为它具有高灵敏度和允许的被分析物的绝对定量。然而，传统的ELISA试剂盒，需要一个比较大的量的样品（一般为100微升），在生物体液中的一些具有高的背景信号，并且被限制为只有一个每2酶联免疫吸附分析物的测量。 最近，一种新的免疫分析技术被引入的情况下，许多这些缺点。此修改后的实验中，由MSD的开发，使用electrochemiluminescENCE（ECL）信号的检测，允许一个非常低的背景和敏感性的增加，能够使用小样本量。电致化学发光是根据记者分子钌（II）trisbipyridal，这是附加的检测抗体。记者分子发射光在620nm时的底部的96 -孔的板，它具有集成在其中3个碳电极的电刺激。另外，通过使用点涂，多个捕获抗体可以被涂覆到一个阱（最多10个96孔板上），允许单个样品中4种不同的蛋白质的同时定量。最近这种技术已被用来衡量促炎细胞因子的血清5,6。从MSD多重板媲美其他多重检测平台7。 我们使用MSD作为主要的试验平台，进一步开发一个定制的复杂板的C同时量化心肌肌球蛋白结合蛋白C（cMyBP-C），肌酸激酶同工酶（CK-MB），心肌肌钙蛋白I（cTnI）的水平进行比较，其结果与monoplex检测的cMyBP C。 CK-MB和cTnI MI行之有效的生物标志物。然而，这些生物标志物的增加可引起心肌梗死以外的其他病症， 如心肌炎或肾功能衰竭8。认为除了增加额外的生物标志物的特异性心肌梗死的诊断。最近，我们已经表明cMyBP-C也是一个潜在的生物标志物MI 9。 cMyBP-C是一种粗丝相关蛋白表达在心脏，10-12，但不是在肌肉骨骼或光滑。因此，循环系统中的cMyBP-C的水平的提高是一个特定的心脏损伤的指标13。 在这项研究中，我们比较了使用自定义复杂的检测与测量血清的cMyBP C uniplex检测，CK-MB cMyBP-C和在MI患者血清肌钙蛋白I。在未来，这种签名技术可以用来诊断心肌梗死的患者，在急诊室出现胸痛。 美国芝加哥Loyola大学的机构审查委员会（IRB）批准了这项研究中使用的人类deidentified样本和使用的免疫法（LU＃20392）。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>MSD Read-Buffer T (4X) with surfactant</td>
			<td>Meso Scale Discovery</td>
			<td>R92TC-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Acros</td>
			<td>9005-64-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MSD Blocker A</td>
			<td>Meso Scale Discovery</td>
			<td>R93BA-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10X</td>
			<td>Fisher BioReagents</td>
			<td>BP399-4</td>
			<td>Filter before use</td>
		</tr>
		<tr>
			<td>Myosin binding protein C antibody, mouse monoclonal (E7), gelatin free</td>
			<td>Santa Cruz</td>
			<td>SC-137180L</td>
			<td>For coating, antibody has to be gelatin free</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Plates and Equipment</td>
		</tr>
		<tr>
			<td>Multi-Array 96-well standard plate</td>
			<td>Meso Scale Discovery</td>
			<td>L15XA-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A prototype 3-plex, four-spot, 96-well plate with cMyBP-C, cTnI and CK-MB</td>
			<td>Meso Scale Discovery</td>
			<td>N452A-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ThermaSeal Sealing Film</td>
			<td>Life Science Products Inc.</td>
			<td>ST-3098</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MSD SECTOR Imager 2400A</td>
			<td>Meso Scale Discovery</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MTS 2/4 digital microtiter shaker</td>
			<td>IKA</td>
			<td>3208001</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a sensitive and specific quantitation method for determination of serum cardiac myosin binding protein-c by electrochemiluminescence immunoassay

生物标志物正变得越来越重要，在临床决策，以及基本的科学。诊断心肌梗死（MI）主要是由患者的血清或血浆中检测心肌特异性蛋白作为心肌损伤的指标。最近的研究表明心肌肌球蛋白结合蛋白C（cMyBP-C）MI后的血清中检测到，我们提出了它作为一种潜在的生物标志物的MI。生物标志物检测通常由传统夹心酶联免疫吸附试验。然而，该技术需要一个大的样品体积，有一个小的动态范围，并且可以测量在一个时间只有一个蛋白质。 在这里，我们示出了复用的免疫测定法，它可以同时测量三个心脏的蛋白质具有很高的灵敏度。测量cMyBP-C在uniplex或共同肌酸激酶MB和肌钙蛋白，我发现类似的敏感性。这种技术使用细观发现（MSD）的复用的方法，在96 - 孔的板，用于检测电致化学发光结合。虽然只有少量样品的要求，实现高灵敏度和大动态范围。使用这种技术，我们测量cMyBP-C，肌酸激酶MB，心肌肌钙蛋白I血清样品从16个科目与MI水平，并与16个对照组比较的结果。我们能够检测这些样品中的所有三个标记和发现所有三种生物标志物来增加心肌梗死后。因此，这种技术是适用于敏感的检测血清样品中的心脏生物标记物的。

3复杂的检测的基本原理和工作流程示于图1，表1中记载的总体工作流程。 uniplex检测沿相同的原理工作的整个底部与捕获抗体以及涂覆除外。信号的检测是通过ECL的电信号被施加到井的底部。这反过来SULFO-TAG标签检测抗体的读缓冲区通过化学反应，将启动一个本地生产的光。这导致了低背景和高灵敏度的检测。在图2中的cMyBP-C在uniplex检测的标准曲线比较的cMyBP中的C 3-plex?...

Watch this Scientific Journal Video about A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay at JoVE.com

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

测量越来越复杂的生物样品中的生物标志物指导临床决策。我们描述一个高度敏感的方法同时测量心肌肌球蛋白结合蛋白C，肌酸激酶MB，心肌肌钙蛋白I与心肌梗死和健康对照组受试者的血清样本。

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

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Research

JoVE Journal

Biology

16.6K 次观看.  Loyola University Chicago. 测量越来越复杂的生物样品中的生物标志物指导临床决策。我们描述一个高度敏感的方法同时测量心肌肌球蛋白结合蛋白C，肌酸激酶MB，心肌肌钙蛋白I与心肌梗死和健康对照组受试者的血清样本。

视频: A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

科研

生物学

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

快速，特异微孔板检测为内和细胞外抗坏血酸在培养细胞的测定

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.