这项研究是由研发项目为国家研究基金会协会（NRF），通过科学，信息和通信技术及未来规划（批准号：2013M3C8A1075908）部资助支持。

1. DNA提取FFPE参考标准细胞系注意：对于这个过程，DNA提取自FFPE参考标准细胞系（HD598，HD593，HD617，HD273和野生型（WT））使用FFPE组织DNA分离试剂盒，如以下方案中所述进行。自动DNA提取按照制造商的总DNA分离指令来实现的。 <ol><li>使用切片机和原始FFPE块，前手工DNA提取和分析准备新鲜的部分，按照既定的程序。确保，所述组织部分（多个）样本的输入分析的不超过10微米的组合总厚度和表面面积不超过600毫米2为一个节。 </li></ol> 1.2 TPS协议注意：在表1所示的量对应处理48个样品所需要的最低限度，并且过程示出的是按照在TPS准则。在实验开始之前沉淀下来的电子管FFPE样品离心600 XG，以避免自动程序过程中样品的损失。 <ol><li>打开自动DNA分离仪和计算机。打开运行控制软件并插入自动加载托盘到TPS甲板装卸区。 </li><li>分配的试剂成其相应的槽，如图1。 <ol><li>放置4个样品（BRAF WT，BRAF V600E 50％，5％和1％）的样品载体机架。 </li><li>将吸头盒在第2和第3的槽，并检查提示每尖端多个过滤器的存在，这将中止运行。 </li><li>确保裂解缓冲液和洗涤缓冲液的适当的混合通过反转他们3-5倍并将它们加载到在塔4（图1）的各个槽。 </li><li>反转的几十倍或轻度vort后exing，加载洗脱缓冲液，磁珠，并在中柱5的小槽的FFPE缓冲器，留下1个时隙空指示的位置（图1， 表1）。 </li><li>加载2ml的深孔板（DWP）到板载体（ 图1）。 </li></ol></li><li>执行以下最后步骤开始运行之前： <ol><li>摘帽所有试管和试剂的低谷。确认足够的容量可在废液瓶。确认固体废弃物酒瓶是空的，内衬生物危害袋。确认该尖端排出板集中在废组件。 </li><li>关闭前盖。 </li></ol></li><li>启动软件。打开NA_Prep_Main_MLSTARlet.med文件。 </li><li>点击“开始”。仪器状态将从空闲切换到运行。 </li><li>输入样本的数目为此运行。选择适合该运行（DNA = 0）所需的方法。输入第一个大体积的位置UME提示，选择“1”，如果所有的盘都满了。进入第一标准体积尖端的位置，选择“1”，如果所有托盘已满。 注：然后，仪器将通过自动化的步骤运行，而无需用户干预。详细的工作流程示于图2，一旦实验结束时，试剂的浪费和尖端注入废物组件。 </li><li>通过使用荧光法定量纯化的基因组DNA。 注意：含有3.3毫微克/微升的最小浓度的DNA样品进行ddPCR分析（第2部分）。 </li></ol> 2. DNA突变剖析：ddPCR协议注意：该协议用于DNA突变谱由三个主要步骤：1）液滴代，2）常规PCR扩增，3）液滴阅读和4）的DNA的突变谱。 2.1。液滴产生注：ddPCR SuperMix混合是推荐ddPCR，因为这个组合中包含所需的墨滴产生试剂。 <ol><li>为了避免污染，遵循标准预防措施，如戴手套，用干净的PCR罩，洁净移液管，低蛋白结合管。 </li><li>确保人类基因组DNA样本的最小浓度为3.3纳克/微升。注意：纯化的DNA试样的浓度的量将基于％突变频率检测的分析而​​变化。 </li><li>装配在96孔PCR板的反应混合物。解冻并平衡反应组分至RT。通过结合2X ddPCR SuperMix混合（10微升）和20引物（正向和反向，900纳米）和探​​头（250纳米）与每个纯化DNA样品（66ng / 2微升）使达到20微升蒸馏水制备PCR反应（如每个液滴发生器协议） </li><li>彻底涡混合以确保均匀和短暂离心分配之前收集在试管底部的内容。装载20＃181，L到墨盒液滴形成。 </li><li>操作液滴发生器，根据制造商的推荐方案<ol><li>将墨盒插入与定位在保持器的上部左侧的盒凹口的保持器。加入20微升的反应混合物含有样品到中间的，和70微升发生器油进入底部的孔中。 </li><li>附上垫片横跨卡盒的顶部。确保垫圈是在支架的两端牢固地钩住;否则，足够的压力为产生液滴将无法实现。 </li><li>按仪器顶部的绿色按钮，打开液滴生成，然后插入。当支架是在正确的位置，这两个电源（左右）和保持器（中间右）指示灯是绿色的。 </li><li>按下顶部的按钮，仪器再次关闭大门，并开始产生液滴。注：按b后utton，歧管自身定位在出口井，通过微流体通道，其中创建液滴拉伸油和样品。水滴流入滴好，在那里积累。液滴指示灯（右）绿色闪烁10秒后，表示液滴产生正在进行中。 </li><li>当液滴生成完成后，所有的3个指示灯变为绿色常亮;通过按下按钮打开车门，并从单位取出固定器。从支架上取下一次性垫圈并丢弃。注意：暗盒的顶端孔中含有液滴，中，下孔几乎空的，用少量的残余油。 </li></ol></li></ol> 2.2。为PCR准备<ol><li>对于每个样品，吸管出40微升从顶部以及墨盒液滴内容插入到推荐的96孔PCR平板的单个以及在制造商的仪器协议指示。否德：使用多通道移液器是理想的转印液滴乳液。水滴缓慢而温和的愿望，建议尽量减少在传输过程中液滴剪切。 </li><li>转移滴，以避免蒸发后，立即密封PCR板带箔。使用可刺穿箔片密封件是与PCR板密封件并在液滴读者针兼容。按照在PCR板封口机使用说明书中的说明。 </li><li>设置板密封温度180℃，时间为5秒。 </li><li>轻触箭头，打开纸盒门。放置支撑块的96孔的一面朝上放在托盘上。将96孔板在载体块，并确保所有板孔与支撑块对齐。 </li><li>用1片材的可刺穿箔密封覆盖96孔板。一旦96孔板被固定在支撑块上，并覆盖上可刺穿箔密封，触摸密封按钮。托盘将关闭和热封将启动。 </li><li>当热密封完成后，门自动打开;从热循环加热块取出盘子，然后取出加热块。 </li><li>确保所有的井板通过检查，在箔洼地超过每显而易见密封良好。密封后，该板块已准备好热循环。 </li><li>一旦液滴被除去，压在盒保持器的闩锁以将其打开。取出空墨盒并将其丢弃。 </li><li>通过以下在表2中详述的参数执行常规PCR扩增。 </li></ol> 2.3微滴读数（根据制造商建议的协议） 注意：继核酸靶的液滴中PCR扩增，液滴读者仪器分析单独使用2色检测系统 13的每个微滴。我们通常设置为检测FAM的ðVIC记者荧光。 <ol><li>点击“刷新系统”素滴阅读器，并使其准备ddPCR分析。 </li><li>板装入液滴阅读器，然后单击“开始”。液滴读者吸取每一个样品，singulates的液滴和液流他们鱼贯过去双色探测器。检测器读出的液滴来枚举阳性和阴性样品。 </li><li>当液滴阅读完毕后，打开车门，从装置中取出板固定器。从支架中取出96孔PCR板和丢弃它。 </li><li>对于适当的保养，更换滴读者油，并根据需要清空废物容器。加入50毫升10％的漂白剂到废液瓶，以防止微生物的生长。 </li></ol> 2.4 DNA的突变分析（按制造商建议的协议） 注意：PCR-阳性和PCR阴性液滴计数，以提供绝对的quantific通货膨胀目标BRAF V600E突变的DNA以数字形式，利用数据分析软件。 <ol><li>单击设置输入有关样品，实验，和实验信息。 </li><li>在设置窗口中，加载板（filename.qlp），然后单击分析以打开和分析数据。数据分析接口被分成三个窗口：结果表，那么选择器选择和处理数据/图形显示。 </li><li>在经处理​​的数据的窗口，对每一个目标浓度数据出现在孔中板图和表列中的结果表。点击集中可视化的浓度曲线的数据。 </li></ol>

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明Ryuri哦，思恩金，和年轻的DEUG金是ABION CRO的员工。

这里，我们强调ddPCR的适用性和DNA分离自FFPE参考标准细胞系样品的特定基因突变的评估。在这项研究中，TPS自动化DNA分离方法用于其可以容易地适应，自动化，并能容纳多达48个不同的样品同时，允许较大规模的实验和较低的变异性。之一，在目前的工作中的DNA分离的局限性是，每FFPE样品是唯一的，并且将变化彼此在表面的污染物，微生物菌群，和/或人的遗传背景。在一般情况下，提取的DNA的质...

基因突变的关键调控基因的累积改变正常细胞的编程细胞样细胞的增殖，分化和存活，从而导致癌症1。在RAS-RAF-MAP激酶途径介导细胞对生长信号。致癌性的BRAF突变可以导致从驱动器中的突变的BRAF基因，这可能会导致BRAF癌蛋白成为过度活跃2。在BRAF基因的突变也导致通过MEK和ERK 3，这反过来，导致的生长因子介导的调控4-6过度细胞生长和增殖独立地过度活跃的下游信号。 有几个工具可以进行DNA突变分析，例如定量实时BRAF V600E突变的福尔马林固定石蜡包埋（FFPE）参考标准细胞株ddPCR。 ddPCR是一种基于PCR的方法，绝对定量相对于传统的定量实时PCR（qPCR的）7,8-提供更高的精度。 ddPCR还提供了更高的分辨率和精度的DNA模板稀有突变的检测，使更多的信息癌症研究和诊断的9。 ddPCR比传统的qPCR的其它优点包括其增强的灵敏度和准确度研究低模板拷贝数10-12时。这里，一个协议，用于自动提取的FFPE参考标准细胞系，接着通过确定BRAF V600E突变由ddPCR存在或不存在的DNA是证明。的软件进行数据分析，分析结果的图形表示的使用也有所说明。整个过程是相对简单且完全取决于样品进行概要的数量和常规PCR和ddPCR机器可用的数量。 以下方案描述的标准程序的BR<html> AF V600E阳性FFPE参考标准细胞系（HD598，HD593，HD617，HD273和野生型（WT））是在一个完全自动化的仪器使用组织制备系统（TPS）协议执行的。随后，分离的DNA样品用于使用ddPCR系统BRAF V600E突变的存在进行分析。进行有针对性的突变分析所有样品已异形之后和数据被加载到数据分析软件。取决于样本的数目/组研究，数据分析，可能需要从一至数小时。该方法的实验部分要求的精度在处理<a href="http://www.jove.com/science-education/5096/isolating-nucleic-acids-from-yeast" title="从酵母中分离核酸，一个朱庇特的科学教育视频解释有关RNA的背景下更多">的DNA</a>和rological移液器和移液器，一朱庇特科学教育视频解释有关移液的上下文中了解更多“&gt;移液到96孔板中，数据分析是用软件来执行时。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>&#160;Hamilton MICROLAB STARlet IVD instrument</td>
			<td>Siemens</td>
			<td>10701001</td>
			<td>Automated DNA isolation instrument</td>
		</tr>
		<tr>
			<td>QX200 Droplet Generator&#160;</td>
			<td>Bio-Rad</td>
			<td>772BR1119</td>
			<td></td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader</td>
			<td>Bio-Rad</td>
			<td>771BR1497</td>
			<td></td>
		</tr>
		<tr>
			<td>Conventional PCR machine capable of ramp-time adjustment</td>
			<td></td>
			<td>621BR17718</td>
			<td></td>
		</tr>
		<tr>
			<td>PX1 PCR plate sealer</td>
			<td>Bio-Rad</td>
			<td>770BR1575</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantaSoft software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA isolation kit&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VERSANT Tissue Preparation Reagents Box 1&#160;</td>
			<td>Siemens</td>
			<td>10632398</td>
			<td></td>
		</tr>
		<tr>
			<td>VERSANT Tissue Preparation Reagents Box 1&#160;</td>
			<td>Siemens</td>
			<td>10632399</td>
			<td></td>
		</tr>
		<tr>
			<td>CO-RE tips</td>
			<td>Siemens</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR mutation analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Supermix&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>BR186-3010</td>
			<td>2X concentration</td>
		</tr>
		<tr>
			<td>DG8 cartridge&#160;</td>
			<td>Bio-Rad&#160;</td>
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employing digital droplet pcr to detect braf v600e mutations in formalin-fixed paraffin-embedded reference standard cell lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

对于我们的ddPCR分析中，我们研究了BRAF V600E突变FFPE参考标准的细胞系。液滴阅读器连接到笔记本电脑上运行的数据分析软件。每个单独的微滴荧光幅度来作为阳性或阴性上定义。由制造商提供的软件还允许输入用户定义的截止定义的正和负的液滴之间的阈值。样品中的正和负液滴的数量用于计算在拷贝/微升术语靶的浓度。 荧光检测和加工成二维散点图显示，定制软件...

Watch this Scientific Journal Video about Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines at JoVE.com

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

该视频的目标是展示如何执行从福尔马林固定的石蜡包埋（FFPE）的参考标准细胞系和数字液滴的PCR（ddPCR）分析的自动化DNA提取，以检测稀有突变在临床环境中。 FFPE样品中检测突变表明ddPCR的FFPE样品中的临床应用。

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

employing-digital-droplet-pcr-to-detect-braf-v600e-mutations-formalin

Research

JoVE Journal

Biology

13.1K 次观看.  Seoul National University. 该视频的目标是展示如何执行从福尔马林固定的石蜡包埋（FFPE）的参考标准细胞系和数字液滴的PCR（ddPCR）分析的自动化DNA提取，以检测稀有突变在临床环境中。 FFPE样品中检测突变表明ddPCR的FFPE样品中的临床应用。 

视频: Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

科研

生物学

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

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 ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

福尔马林固定石蜡包埋的组织核心的制备RNA和DNA提取

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

We describe the use of quantitative immunoblotting to validate immunofluorescence histology coupled with image analysis as a means of quantifying a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Our results demonstrate the utility of immunofluorescence histology for ascertaining the relative quantity of biomarker proteins in routine biopsy samples.

细胞系定量免疫印迹作为常规组织样本中生物标志物蛋白定量免疫荧光定量的标准

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

用于SARS-CoV-2检测和定量的两步逆转录液滴数字PCR方案

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...