从心理健康国家研究所：描述该项目由奖号码R01MH079751（F.佩鲁齐PI）的支持。内容完全是作者的责任，并不一定代表心理健康国家研究所和美国国立卫生研究院的官方意见。

以下协议描述了标准程序，从使用现成PCR板CSF和轮廓的microRNA分离RNA。需要注意的是，有机相萃取是可选的，并且该提取的RNA以确保最大的恢复过程中的载体RNA被加入到样品中。因此，没有必要进行量化的RNA。 总体而言，平均为6-8板可以在一天之内运行，如果该cDNA之前（约2小时）的天合成。当所有的样品已经成型并加载到软件进行数据分析。取决于样品/组或它们的组合的用于比较的数量，数据分析可能需要从一个到几个小时。 1。 RNA提取的RNA提取从脑脊液样品上进行，冷冻储存在-80℃下以等分试样直至分析。用于此过程的mirVana巴黎分离试剂盒被使用时，按照制造商的仪器ctions总RNA分离。请注意，虽然富集小RNA种类可以使用该试剂盒，该步骤不进行和RNA的提取步骤的总RNA分离步骤之后结束。此外，虽然mirVana巴黎分离试剂盒（像其他市售的RNA提取试剂盒）不需要有机萃取，协议对本程序可以在下面找到。 的协议，用于RNA提取由两个步骤组成： 1.1。有机萃取（如果没有使用mirVana巴黎RNA提取试剂盒必填） 1.2.2。总RNA的提取 1.1。有机萃取 1.1.1。制备试剂<ol><li>添加375微升2 - 巯基乙醇的2倍变性溶液中，并将其储存于4℃。 </li></ol> 1.1.2。准备脑脊液<ol><li>在RNA提取前解冻CSF对冰每等分。添加1 MS2 RNA载体微克至0.5毫升解冻脑脊液，轻轻混合。请注意，如果省略了有机提取，对MS2载体将被添加到在1.2.2.1下面描述的样本之前的RNA分离。 </li><li>混合0.5毫升2倍变性溶液在室温至脑脊液。 </li><li>加入1 ml酸 - 苯酚：氯仿CSF被加上2倍的变性溶液：一定要取出含磺酸 - 苯酚的底部相：氯仿，并不在于对混合物的顶部的水性缓冲液。另外，请注意，酸酚：氯仿含有苯酚，这一试剂工作时，这是一种毒药和刺激性，使用手套和其他个人防护装备。 </li><li>旋涡30-60秒混匀。为方便起见，2毫升样品/变性溶液/酸 - 苯酚：氯仿混合物分成2个1.5毫升的Eppendorf管中。 </li><li>在室温下离心以最大速度5分钟（10,000 XG）。 </li><li>小心除去水（上部）相，而不会干扰下层相或相间并将其传输到一个新管。注意恢复音量。 </li></ol> 1.2。 RNA分离我们建议有一个专门的长凳上，设置移液器，和机架处理RNA样品。工作区域和工具使用RNA酶扎普喷雾是净化和湿巾前每个实验。 1.2.1。制备试剂： <ol><li>加21毫升100％乙醇中，miRNA的洗涤液1和40毫升100％乙醇洗涤溶液的2/3。请注意，miRNA的清洗液1包含硫氰酸胍是一种潜在的危险物质。 </li></ol> 1.2.2。总RNA的提取<ol><li>添加1.25体积室温的100％乙醇，从有机萃取水相，并充分混合。 </li><li>将滤芯放入收集管中的一个。 </li><li>吸管不超过700微升裂解液/乙醇混合物到滤芯。 </li><li>离心30秒在最大速度。应用超过700微升在连续应用到相同的滤波器的混合物。每次离心后弃去流通和保存收集管的清洗步骤。 </li><li>适用于700微升的miRNA清洗液1的滤芯，离心15秒，最大速度。通过从收集管中丢弃的流量和更换滤芯到同一个收集管。 </li><li>适用于500微升洗涤液的2/3，离心15秒以最大速度。通过从收集管中丢弃的流量和更换滤芯到同一个收集管。申请第二个500微升洗涤液的2/3，离心15秒，最大速度。通过从收集管中丢弃的流量和更换滤芯到同一个收集管。旋转组件1分钟以最大速度从过滤器除去残留的液体。 </li><li>转移滤芯到一个新的收集管。适用于100＆万亩;升的预热（95℃）无核酸酶的水至过滤器的中心，并关闭盖子。离心30秒以最大速度恢复的RNA。 </li><li>收集的洗脱液中含有的RNA，并将其储存于-80℃用于后续的应用。 </li><li>量化1μlRNA提取的使用NanoDrop 2000c的分光光度计。需要注意的是RNA定量可以如果一种RNA载体的RNA提取过程中加入到样品中被跳过。在这种情况下，8μL的RNA受到cDNA合成，详见下文。 </li></ol> 2。 miRNA的个人资料：定量RT-PCR检测协议该协议为miRNA谱由两个步骤组成： <ol><li>第一链cDNA合成</li><li>实时PCR扩增</li></ol> 2.1。第一链cDNA合成 2.1.1。稀释模板的RNA <ol><li>每个模板RNA样品调整至5纳克/微升使用不含核酸酶的水的浓度。如果RNA并没有被量化（见步骤1.2.2.9），然后8μL的RNA被用于cDNA合成</li></ol> 2.1.2。制备试剂<ol><li>轻轻解冻5×反应缓冲液和无核酸酶的水，并立即放置冰上。 </li><li>涡旋混匀。重悬的RNA穗中加入40μL无核酸酶的水到管涡旋混合，让它在冰上15-20分钟。临用前，从冰箱中取出酶混合物，轻弹管混合并放置冰上。降速所有试剂。 </li></ol> 2.1.3。逆转录反应设置<ol><li>准备所需的1.5ml离心管，或同等RT工作液量（以比例如表1所示，除RNA模板），涡旋（1-2秒，最大速度）混匀，短暂降速（我们使用一个小型的Eppendorf离心机与固定测速，10秒），并将其放置在冰上。注意，UniSp6核糖核酸穗-在之前的RT反应加入到样品（ 见表1）。 </li><li>免除RT工作液为无核酸酶PCR管。 </li><li>分配模板RNA在每个管中。 注：记得要计算10％的过量体积/每个试剂的移液和/或机械死体积。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂 </td><td> 面板I体积（μl） </td><td> 面板I + II体积（μl） </td></tr><tr><td> 5X反应缓冲液</td><td> 4.4 </td><td> 8.8 </td></tr><tr><td>无核酸酶的水</td><td> 9.9 </td><td> 19.8 </td></tr><tr><td>酶混合物</td><td> 2.2 </td><td> 4.4 </td></tr><tr><td> UniSp6 RNA穗模板</td><td> 1.1 </td><td> 2.2 </td></tr><tr><td>模板总RNA（5纳克/微升） </td><td> 4.4 </td><td> 8.8 </td></tř&gt; <tr><td> 总成交量 </td><td> 22 </td><td> 44 </td></tr></tbody></table> 表1。逆转录反应设置。 </li></ol> 2.1.4。混合和自旋试剂<ol><li>混合通过温和（1-2秒）涡旋的反应，以确保所有试剂充分混合。混合后，短暂离心（微型Eppendorf离心机以固定的速度，10秒）。 </li></ol> 2.1.5。培育和热失活<ol><li>程序PCR仪如下： </li><li>孵育60分钟，在42℃下</li><li>加热灭活在95℃下的逆转录酶5分钟</li><li>立即冷却至4℃ </li><li>储存于4°C或冻结 注意：该协议可以在这个阶段被中断。未稀释的cDNA可保持在-20℃下长达5周（在4℃可选存储长达4天）。 </li></ol><p类=“jove_step”&gt; 2.2。实时PCR扩增 2.2.1。准备试剂，用于实时定量PCR <ol><li>冰上放置的cDNA（从步骤2.1.5）。 </li><li>解冻无核酸酶的水和PCR扩增预混冰上。用铝箔覆盖它们避光PCR扩增预混试剂瓶。临用前，用涡旋1-2秒，迷你离心机混合PCR扩增预混和自旋向下在1500×g离心1分钟。 </li></ol> 2.2.2。结合SYBR Green母液，水，和cDNA，并加入到PCR板下面的过程，建议，以避免粘到管表面低浓度的cDNA： <ol><li>在取出板密封，在短暂冷冻离心机降速板（S）在1500×g离心1分钟。 </li><li>在一个15毫升的锥形管，结合2X PCR扩增预混试剂和水。第一小组：2,000微升2个主结构和1,980μl水;面板I + II：4,000微升2个主结构和3,960μ;升水。 </li><li>加入20微升的cDNA（面板I）或40μL的cDNA（面板I + II）和混合。 </li><li>涡旋1-2秒，轻轻混匀，并在冷冻离心机降速为1500×g离心1分钟。 </li><li>将PCR扩增预混/基因组合，多道移液器水库。可能必须识别储层具有多通道移液管的长度。这样可以使体积的足够高的允许等体积的等分试样在整个多声道的电平。这是对过去的几等份的关键。 </li><li>分配10μl的PCR主混合/混合的cDNA至384 - 孔板的使用16通道移液器各孔中。 </li><li>与光学密封密封板。 </li><li>在冷冻离心机（1500×g离心1分钟）旋转简要板，以混合溶液并除去气泡。 注意：本实验可以在此时被暂停。存储避光，在4℃下进行长达24小时的反应。 </li></ol> 2.2.3。实时时间PCR 以下在表2中详述的参数进行实时PCR扩增和熔解曲线分析。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> 流程步骤 </td><td> 设置，罗氏LC480 </td></tr><tr><td>聚合酶活化/变性</td><td> 95℃，10分钟</td></tr><tr><td>放大</td><td>扩增45个循环; 95°C，10秒 60℃，1分钟 升温速率 1.6°C /秒 光学读</td></tr><tr><td>熔解曲线分析</td><td>是的</td></tr></tbody></table> 表2。使用Roche的LightCycler 480实时荧光定量PCR循环条件。 2.3。实时荧光定量PCR数据分析实时PCR数据分析是完成与Exiqon GENEX专业5.0，下面的T他建议说明。该GENEX一步一步的手册可在下载<a href="http://www.exiqon.com/ls/Documents/Scientific/Exiqon-data-analysis-guide.pdf">http://www.exiqon.com/ls/Documents/Scientific/Exiqon-data-analysis-guide.pdf</a> 数据分析包括三个步骤： <ol><li>数据的导入</li><li>数据的预处理</li><li>统计分析</li></ol> 2.3.1。数据的导入<ol><li>在罗氏公司的LightCycler 480选择第二衍生物的分析方法和计算CQ值。数据导出为一个表，并保存为文本文件。 </li><li>要导入数据，开放GENEX并点击Exiqon导入向导按钮，然后点击“开始”。按照说明来选择格式，仪表板和布局文件（S）。需要注意的是板布局文件（Excel文件）从Exiqon网站（被下载<a href="http://www.exiqon.com/plate-layout-files">http://www.exiqon.com/plate-layout-files</a> ）。 </li><li> IMPOR吨面板I和II，并单击“下一步”。 </li><li>文件导入后生成的表包含预定义的列。样品名称可以被编辑和分类的列可以被添加或在该步骤中除去。点击“下一步”，当你这样做。 </li><li>保存数据并加载到数据编辑器。 </li></ol> 2.3.2。数据的预处理<ol><li>所推荐的Exiqon和GENEX，比较多个板时，要做的第一件事就是从预处理菜单中选择板间的校准来校准板之间的数据。 </li><li>建议在一式三份的运行运行的miRNA阵列。如果一个miRNA的不存在，至少在三分之二的副本将被设置为nonexpressed。 </li><li>在预处理的下一个步骤中，设置一个隔断。限定切断值指示与阈值循环（C T），高于此值的数据将被丢弃。在使用脑脊液标本，本实验中，切断设定在39。疗法安伏，用C T比三个副本39更高的所有样本将被丢弃。如果与miRNA具有C T&gt; 39为一个探针（一个复制品出三个），该读数将与在C t的另外两个探针的平均取代，条件是它们均低于39。 </li><li>定义参考基因。 GENEX专业有利用geNorm和/或NormFinder识别参照基因的选择。选择具有类似CQ值在所有样品并运行geNorm和/或NormFinder miRNA的列表。根据这样的分析，在这里提供的例子中，miR-622和miR-1266具有横跨试样的最均匀的值，并且被选择作为参考基因（ 图1）。 </li><li>接着，数据使用的参考基因归一化并且所获得的值对应于ΔC 吨 </li><li>如果cDNA合成反应中以一式三份进行，在这点的值进行平均。 </li><li>验证表重新移动空，几乎空列。在预处理的窗口中选择“验证表”，并比对申请删除空列线。在下面的行，选择有效数据所需的百分比，然后单击应用。例如，选择100％，如果你想比较通用于所有样品只的miRNA。 </li><li>处理缺失数据。 请从预处理菜单中的“缺失数据”并选择选项来处理丢失的数据之一。此步骤是必需的。请注意，如果您尝试加载包含未处理丢失的数据文件GENEX会给你错误。 </li><li>确定样本（ 即 。实验与对照）组之间相对定量。群体之间的相对定量是使用ΔΔC T法计算。在GENEX，单击预处理选项卡，并选择相对定量。在窗口中选择参照组和命中适用。请注意，对于图形表示和/或作进一步的统计分析中，expr分裂国家的数据应该转换为对数刻度。在预处理菜单中，选择LOG2。 </li></ol> 2.3.3。统计分析 GENEX软件允许多种统计分析，包括t检验和方差分析。 <ol><li>加载最终mdf文件到GENEX和开放的数据管理。关键是要选择其中进行统计分析的样品的正确的样品或组。 </li><li>选择“统计”，并选择对应于所期望的测试的图标。 </li><li>单击“运行”，在控制面板窗口。结果保存为Excel或MDF文件。 </li></ol> 2.3.4。表达谱小分子RNA和样品可分类，聚类和可视化的热图和树状图，如下<ol><li>加载最终mdf文件到GENEX和开放的数据管理。使用此窗口来选择并创建样品或miRNA的群体。单击“应用”时，你就完成了。 </li> &lt;LI&gt;在左上角选择群集，然后单击上的热图图标</li><li>在控制面板窗口中，单击“运行”。热图可以储存在不同的格式，如TIFF或BMP，它也可以被复制，并根据需要进行修改。 </li></ol>

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作者什么都没有透露。

microRNA是小非编码RNA，通过抑制翻译和/或促进mRNA降解2调节基因表达。由于其高稳定性在无细胞的条件下，微小RNA已在许多体液检测。此外，microRNA的独特表达谱已与相关阶段和/或进展中的各种人类癌症3-9。 有可用来分析小分子RNA的几种工具，其中包括经典的芯片阵列或最新的深度测序技术。然而，我们选择利用一个高度敏感的qPCR平台10，11，其具有...

小分子RNA属于小型的家庭（21-23核苷酸长）非编码调控基因表达转录后的RNA。微小RNA可分泌在它们似乎是相对稳定的细胞外空间。在确定改变miRNA的表达可以向鉴定生物标志物的一个重要步骤，进行miRNA表达谱和处理大量数据的可吓人。 在这里，我们描述了一种协议，以确定在脑脊液（适用于其他体液）由一个敏感的实时PCR的变化的miRNA表达。我们还描述了使用软件进行数据分析，包括统计分析和结果的图形表示。整个过程是比较容易和简单，并根据样本进行概要分析和实时PCR机器的数量可用，也比较快的数目。实验部分要求精度处理RNA和移液到384孔板，同时采用GENEX数据分析部分要求在信息学和统计学的一些基本知识。

Table III. List of equipment
<table border="1" cellpadding="0" cellspacing="0" style="width:655px;" width="655">
	<tbody>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:27px;">
			Equipment
			</td>
			<td nowrap="nowrap" style="width:150px;height:27px;">
			Company
			</td>
			<td nowrap="nowrap" style="width:204px;height:27px;">
			Catalog Number
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Finnpipette Novus Multichannel Pipetter
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Thermo Scientific
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			HH05279
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Finntip Pipette Tips
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Thermo Scientific
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			9400613
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Thermal Cycler C1000
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Biorad
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			0.2 ml Low Profile, Clear PCR tubes
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Biorad
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			TLS0801
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Flat Cap Strips
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Biorad
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			TLS0803
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			LightCycler&#174; 480 Real-Time PCR System
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Roche
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			LightCycler&#174; 480 Sealing Foil
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Roche
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			04 729 757 001
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Refrigerated Centrifuge 5804R
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Eppendorf
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Swing-bucket Rotor, 4-place
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Eppendorf
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			A-4-44
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Bench-Top Mini Centrifuge
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			05-090-100
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Bench-Top Vortex
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			1.5 ml Microcentrifuge Tubes, Sterilized
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			02-681-5
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Matrix Reagent Reservoir, 25 ml
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Thermo Scientific
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			8093
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Thermomixer Confort
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Eppendorf
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Bench-To Mini Centrifuge Sigma 1-15
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Sigma
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Bench-Top Vortex
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Velp Scientifica
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			F202A0173
			</td>
		</tr>
	</tbody>
</table>

Table IV. List of reagents
<table border="1" cellpadding="0" cellspacing="0" style="width:655px;" width="655">
	<tbody>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:25px;">
			Reagents
			</td>
			<td nowrap="nowrap" style="width:168px;height:25px;">
			Company
			</td>
			<td nowrap="nowrap" style="width:144px;height:25px;">
			Catalog Number
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			Universal c-DNA synthesis kit, 16-32 rxns
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Exiqon
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			203300
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			SYBR Green master mix, Universal RT, 25 ml
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Exiqon
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			203400
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			Ultrapure Distilled Water Dnase, Rnase free
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Invitrogen
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			10977-015
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			MicroRNA Ready to use PCR Human Panels (I+II) V2.R
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Exiqon
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			203608
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			mirVana&#8482; miRNA isolation Kit, 40 isolations
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Ambion
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			AM1556
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			MS2 RNA carrier
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Roche Applied Science
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			10165948001
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			2Betamercaptoethanol 98%
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Sigma Aldrich
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			M3148-25ml
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			Ethanol 100%
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Carlo Erba
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			64-17-5
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			Nuclease free water
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Qiagen
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			1039480
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			RNAse Zap&#174;
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Ambion
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			AM9780
			</td>
		</tr>
	</tbody>
</table>

cerebrospinal fluid microrna profiling using quantitative real time pcr

<html>微RNA（miRNA）构成由指导RISC到目标位于mRNA的位点，因此，通过调节它们的翻译抑制基因调控的一种有效层。改变的miRNA表达已被证明参与所有主要复杂疾病的发展。此外，最近的研究结果表明，miRNA能够被分泌到细胞外环境，并进入血液和其他体液中，他们可以以高稳定性循环。这种循环miRNA的功能在很大程度上仍然是难以捉摸的，但系统的高通量方法，如miRNA表达谱阵列，已经导致miRNA的签名鉴定的几个病理条件下，包括神经退行性疾病和多种类型的癌症。在此背景下，miRNA的表达谱在脑脊液鉴定，据报道在我们最近的研究，使得miRNA的有吸引力的候选生物标志物分析。 _content“&gt;有可用于分析小分子RNA，如微阵列几个工具，定量实时PCR（定量PCR），以及深度测序。在这里，我们描述一个敏感的方法，通过实时定量PCR技术来分析在脑脊髓液的microRNA。我们用Exiqon的microRNA准备使用的PCR人类面板I和II V2.R，它允许检测742独特的人类微小RNA，我们进行的阵列，一式三份运行，我们使用GENEX专业5软件进行处理和分析数据。 使用该协议，我们已经成功地描出的microRNA在各种类型的细胞系和原代细胞，脑脊髓液，血浆和福尔马林固定，石蜡包埋的组织。

从本研究结果先前已公布的1。 图1显示了使用geNorm应用候选参考microRNA的分析结果。因此，2微小RNA中，miR-622和miR-1266，被确定为参考基因和被用于归一化的miRNA值。 对于脑脊液研究中，我们有三组样品的HIV-（N = 10），HIVE（N = 4）和HIV（+）无脑炎（HIV（+）中，n = 5）。这两个群体，HIVE和HIV阳性，与对照病毒组进行了比较。经统计分析?...

Watch this Scientific Journal Video about Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR at JoVE.com

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

我们描述了实时PCR的协议来分析小分子RNA在脑脊液（CSF）。除的RNA提取的协议，该过程可以扩展到从其它体液，细胞培养，或组织标本中提取的RNA。

脑脊液的microRNA分析采用实时定量PCR技术

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

cerebrospinal-fluid-microrna-profiling-using-quantitative-real-time

Research

JoVE Journal

Medicine

15.3K 次观看.  LSU Health Sciences Center. 我们描述了实时PCR的协议来分析小分子RNA在脑脊液（CSF）。除的RNA提取的协议，该过程可以扩展到从其它体液，细胞培养，或组织标本中提取的RNA。 

视频: Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

科研

医学

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

的运动再培训采用实时反馈的性能

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

实时的细胞毒性分析在人全血

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

在新生小鼠肺肺流体体积的实时X射线成像

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

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

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

分离和剖析人力胆汁含外来体的MicroRNA-

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

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...