Name: perturbations of circulating mirnas in irritable bowel syndrome detected using a multiplexed high-throughput gene expression platform
Uploaded: 2016-11-30T13:00:00.000+00:00
Duration: 10 min 37 s
Description: Watch this Scientific Journal Video about Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform at JoVE.com

The authors acknowledge funding from the U.S. Department of Health and Human Services, the National Institutes of Health, the National Institute of Nursing Research, and the Division of Intramural Research to Wendy A. Henderson, 1ZIANR000018-01-06; the Intramural Research Training Awards to Nicolaas H. Fourie, Ralph M. Peace, Sarah K. Abey, and Leeanne B. Sherwin; and special support from the Burroughs Wellcome Fund to Howard Hughes-NIH Research Scholar Ralph M. Peace.
The opinions expressed herein and the interpretation and reporting of these data are the responsibility of the author(s) and should not be seen as an official recommendation, interpretation, or policy of the National Institutes of Health or the United States Government.

这里所描述的研究是经国家卫生研究院的机构审查委员会（Clinicaltrial.gov＃NCT00824941）。 1.全血样本的收集<ol><li>通过在含有RNA稳定溶液（允许长期储存）血液收集管静脉穿刺收集来自研究参与者的全血样品（2.5毫升）中，并将其存储在-80℃直至RNA纯化。 </li></ol> 2.总RNA纯化<ol><li>解冻并培育血液试管2小时，然后在5,000×用摇出转子10分钟离心它们克通过仔细倾倒或吹打它关闭到废液管，小心不要打扰颗粒去除上清。 </li><li>加入4毫升无RNase的水洗涤沉淀，安全地更换帽，和旋涡直到沉淀溶解明显。离心机利用摆动斗转子以5,000×g离心10分钟，再移动上清液，如在步骤2.1中描述。 </li><li>加入350微升BM1缓冲液（提供）来沉淀。涡旋沉淀直至其明显溶解并转移到用于自动提取总RNA一个2毫升的加工管。 </li><li>执行细胞内的RNA，包括的miRNA的自动化纯化，从用市售RNA提取试剂盒，其可以机器人RNA提取系统上进行自动化的全血。 注意:我们使用的自动化机器人系统可以同时处理12个抽样，并花费3小时的RNA纯化协议来完成。 <ol><li>加载预加载枪头，离心管，缓冲剂，并在RNA纯化试剂盒提供到机器人RNA提取系统的试剂。 </li><li>从协议选择菜单中的"血的miRNA部分A"协议，并开始运行。 注:的自动化过程的描述见补充材料。 </li><li>一旦机器人合作自动化的miRNA纯化方案mpleted A部分，清空废容器和机器人系统中删除所使用的塑料和试剂容器。 </li><li>关闭所有含洗脱RNA微离心管盖子，并将其放置在摇床适配器。从协议选择菜单中选择"血的miRNA部分B"在65℃至孵育样品5分钟。 </li><li>一旦机器人已完成运行的miRNA纯化方案的B部分，立即取出样本，他们寒气冰。 </li></ol></li><li>定量和使用分光光度计评估纯化的RNA质量。 注意:根据为所述miRNA测定中，33纳克的最低浓度的协议建议/微升是必需的，所有样本应满足或超过一个二百六十零分之二百八十○比率为1.9和最小260/230比率为1.8，以确保不存在显著有机污染物，这可能会影响测定性能（参照附图17）。 </li><li>在-80 摄氏度保存样本</li></ol> 3. miRNA的样品制备<ol><li>正常化12 RNA样品至33毫微克/微升使用不含核酸酶的水。 </li><li>制备1:500稀释使用不含核酸酶的水的测定试剂盒中提供的miRNA的控制。置于冰上。 </li><li>准备退火主结构:采用10和20微升移液器结合13微升退火缓冲液（提供），26微升miRtag试剂（提供），和6.5微升的miRNA控制（步骤3.2制备）。 </li><li>使用10微升吸管，添加退火预混3.5微升3微升RNA样品（ 即 100毫微克），以每十0.2毫升条管的。弗里克混合，降速在热循环使用台式微型离心（2000 XG）和地点（94℃下1分钟，65℃下2分钟，10分钟，45℃;保持在48℃） 。 </li><li>结合3μ准备结扎预混使用20微升吸管;（提供PEG）（提供）的连接缓冲液的升和19.5微升聚乙二醇的。使用10微升吸管添加2.5微升连接主混合物的每个条管从步骤3.4。 <ol><li>轻轻混匀，降速使用台式微型离心（2000 XG），并在48 摄氏度回热循环5分钟。 5分钟后，用10微升吸管，在热循环仪在48℃下加入1微升连接酶直接到每个试管中，并培育他们3分钟，47℃下3分钟，46℃下3分钟，45 度下进行5分钟，和65℃下10分钟;保持在4℃ </li></ol></li><li>从热循环仪取出试管，将它们轻轻混匀，使用台式微型离心（2000 XG）旋转下来，并添加1μl连接清理酶（提供）。孵育管在热循环（37℃1小时，10分钟，70℃;保持在4° </s达&gt; C）。用10微升吸管。 </li><li>取出管，并用100微升吸管，加入40微升无核酸酶水，混合，降速使用台式微型离心（2000 XG）。 </li></ol> 4. miRNA的杂交<ol><li>在冰上解冻记者与捕获探针集（提供），并使用台式微型离心（2000 XG）旋转下来。 </li><li>使用200微升移液器，添加130微升杂交缓冲液的记者代码集管（130微升）创建杂交预混。混合和降速使用台式微型离心（2000 XG）。 注:记者和捕获探针是特异性和标准为每个miRNA包含在面板中。定制面板和用户设计的捕获和探针组可设计。 </li><li>在12个新的0.2毫升的条管，使用20微升移液器加入20微升杂交预混。 </li><li>变性步骤3.7 miRNA的样品在85 摄氏度 FOR 5分钟，冷却，冰浴。使用10微升吸管添加5微升至每个管从步骤4.3。 </li><li>计划在热循环到65℃时的30μl体积计算的时间永远温度和热盖设置（没有编程它在运行结束递减至4℃）。 </li><li>使用10微升移液管，加设各管捕获探针的5微升，混匀，降速使用台式微型离心（2000 XG），并立即放入热循环在65℃。孵育不低于12小时，不超过30小时。 </li></ol> 5.准备站和数字分析仪<ol><li>装入准备站。 <ol><li>打开站门并装入试剂板（提供），盒（提供），枪头（提供），十二0.2毫升的条管和十二个空0.2毫升条管制备样品（提供）。取下带管帽和试剂板盖。关闭准备站门并运行从控制面板的相应检测。 注:杂交后的处理是一样的，无论是检测正在运行的。所有试剂和塑料制品是预包装的，不需要准备，比使它们至室温，并在2000 xg离心纺丝向下试剂含有96孔板2分钟其他。所有组件都装在准备站明确的立场。见补充材料的自动化程序的描述。 </li></ol></li><li>可视化和算上固定和面向三方配合的条形码。 <ol><li>从准备站中取出墨盒，所提供的盖玻片密封它，并将其放置在相机光学上方的插槽数字分析仪。 </li><li>选择适当的测定法（miRNA的面板测定）和测定版本的测定试剂盒表示，并且运行该程序。 注:数字分析然后采取视点图像领域对每个样品位置吨4激发波长，允许该荧光条形码被读取和解码。特定条形码，对应于一个特定的miRNA，进行计数。 </li><li>经由因特网链接数据文件导出到USB或直接到服务器。 </li></ol></li></ol> 6.数据处理与分析<ol><li>点击"原始数据"选项卡，选择"导入RCC文件"。从USB含RCC文件（原始数据文件）的文件夹。 </li><li>点击"下一步"，选择所有QC框，然后点击"导入"。 </li><li>点击"新探"选项卡。命名和描述的研究和保存。 </li><li>在新的研究中，选择"新实验"标签和名称，描述了新的实验。点击"下一步"，选择从包含已上传相关的原始数据文件左侧窗格中的RLF文件夹。一旦它被选定后，点击"继续选择"，然后单击"下一步"。 </li><li>加注释如果需要的话，然后单击"下一步"。 </li><li>选择背景减除法;任一阴性对照空白车道减法，或用户定义的减法可以选择。一旦选中，单击"下一步"。 注:确定基于所述技术参数和研究的生物上下文中最适合的背景校正的方法。 注:计数是低丰度的目标不太精确。对于我们在这里描述了研究的目的，首先是没有背景校正，以检查质量控制参数，并检查计数跨技术复制不同的丰度的miRNA的变化处理的数据。然后，数据是使用25计数背景阈重新处理。 </li><li>选择"百强"正常化，然后单击"下一步"。 </li><li>为了获得比数据，选择比参数，然后单击"下一步"。 </li><li>点击"完成"。 </li><li>点击名为恩periment在左窗格中露出一个下拉菜单，里面包含"原"，"标准化"，"分组"，"比数据"和"分析数据"。 </li><li>点击"原始数据"，并观察右窗格中的QC报告。观察旁边不传递默认的QC参数样品的标志。建立一个新的"学习"，这不包括标记的样品并如所述重新处理，或者简单地通过选择"排除由QC /正常化标志样品"排除从数据分析步骤被标记的QC样品。 <ol><li>出口"原料"，"正常化"，或CVS"群"的数据文件，或在其他统计或微阵列软件进行分析互相兼容的文件格式。 </li></ol></li><li>从左侧窗格中选择"归一化数据"，然后选择要包含在右侧窗格中的分析样本。 </li><li>点击"分析"，然后选择所需的分析类型（ 例如，"热图＆＃34 ;,"小提琴情节"，"箱线图"，"散点图"或"直方图"）。选择所需的排除标准盒（ 例如，排除离群样品或基因，由QC标志排除的样品中，排除在控制基因，和/或排除从数据分析正常化探针）。 </li><li>选择个别样品从分析根据需要被排除。 </li><li>选择单个基因要从排除在外，或根据需要纳入分析。 </li><li>选择所选的统计和数据可视化的操作参数，然后单击"完成"。 </li></ol>

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W.A.H., S.K.A, N.H.F., and R.M.P. hold a US patent (no. PCT/US14/38638) for a circulating miRNA signature of IBS emanating from the research described herein.

该议定书中的关键步骤 特别是对于所述miRNA测定中，关键是不使用未纯化的裂解物（这些可以在mRNA和蛋白测定中使用的）中，作为试剂尚未用于裂解物进行了优化，和样品制备的反应很可能被抑制。此外，污染物从裂解和RNA纯化步骤（ 例如，胍化合物，乙醇和酚类）结转可抑制结扎和样品纯化的反应。良好品质的RNA因此与miRNA测定的灵敏度和准确度的关键。 使用热循环与加热的盖是至关重要的，以确保适当的温度控制，以优化的所有反应步骤的性能。在步骤3.5.1，它不以除去由热循环条，以便在加入连接酶的过程中，保持温度是重要的。除去条带以添加连接酶将损害该反应。当执行杂交步骤，重要的是，避免剧烈摇动，移液，或轻弹在管混合试剂时，因为这会剪切报告探针。为了确保最佳的性能和一致性，通过温和轻弹调匀在管试剂是重要的。总是降速反应混合后，并使用新的移液管每次试剂分注以确保准确的吸液和，以避免意外的交叉污染时翻倒。 修改和故障排除 可定制的代码集，包括感兴趣的只有目标。以这种方式，成本可以平衡，以允许对大量样品的检测时进一步调查或验证生物签名。自定义探针可设计为基因或目标不能从菜单点菜 。灵敏度丰裕少目标或低浓度的RNA可以通过允许更长的杂交时间和/或通过使用较高分辨率的数字计数操作。 在我们的研究中，我们使用，从全血中的总RNA预定的800靶miRNA面板;但是，可以自定义的试验，包括更少的miRNA如果需要更有针对性的做法。共有24个样品可在一天制备，在两组12的交错，因为两个盒可以舒适地通过杂交后处理机器人准备站上通过，并在第二天对数字分析仪成像。在12批次样品的交错处理重要的是要规范杂交时间。已拍摄墨盒可以保存并在 4℃将重新扫描存储的数据是否分析认为，较高的分辨率扫描可能会提高罕见的miRNA的检测。罕见分子的检测也可通过为更长的杂交样品改善;然而，长期的杂交可能resul吨过饱和，并且可以仅提高的结果，如果在开始的RNA浓度低（如在细胞外囊泡衍生的RNA）。该平台的灵敏度和精确度允许相对低丰度的miRNA，以可靠地计量。 该技术的局限性 所描述的基因表达分析平台只可容纳高达每组800目标。因此，它不适合整体的miRNA转录/全转录组的研究。唯一已知的，描述的，并且可以使用平台来检测指定的目标，因此，它不适合于新的分子物种的发现。其它方法，如RNA测序或其他传统的微阵列的方法，更适合于全转录探索性研究。 关于到现有/替代方法的技术意义 IBS的特征在于症状的组合，首席cipally包括腹痛;内脏敏感性;和大便习惯，如频繁腹泻，便秘，或两者9,10的组合的变化。 IBS症状没有已知的器质性原因，和病人不与临床显著炎症，胃肠组织，或全身性标志物9-12的病理扰动出现。研究表明，在IBS生物失调是潜移默化的，亚临床和异类，与新兴的共同主题周围炎症和免疫功能的10。因此，我们需要控制实验者引入变化，并使用了一个平台，是能够在基因表达可靠地检测细微扰动。试验和系统的独特属性规范来样加工，减少实验者通过自动化处理，减少了技术差异产生高度可重复的数据。这些特点保证了重点调查，并生产高精密和Replicable数据。这允许检测可能由更丰富的目标的信号被忽略或淹没低表达目标之间微妙扰动和扰动的使用强度 - 或扩增为基础的方法时。基于PCR的微阵列和RNA测序方法是可行的替代方案使用所描述的平台和当需要更高的吞吐量，或者可以是作为替代的吸引力，当目的是表征全转录或以寻找新的分子。然而，替代品可能无法准确，灵敏地检测和定量稀有的目标和微妙的扰动进行为好。 掌握这一技术后，未来的应用方向或 在IBS参与者循环miRNA表达呈数目被发现是既显著和类别内是一致的扰动。该鉴定的miRNA与相关通道法相关YS和病理状况，包括功能性疼痛病症（的miR-342-3p:慢性膀胱疼痛）8和炎症（的miR-150）13。本例研究是基于用于定义目标和利益的系统的探索队列。然后，更有针对性的，更小的miRNA阵列构造，降低了每个样品的成本并允许更大的同伙以符合成本效益的方式，以验证和优化的签名和生物标志物进行分析。基因表达平台测定系统撞击微阵列的传统探索性质和更有针对性，假设驱动的数据收集来优化和测试分子签名和生物标记14-16之间的平衡。该方法将被用于检查分子和蛋白质的扰动在体内和体外模型，其中所述靶miRNA是实验过表达或抑制。新测定法允许的mRNA和p的同时定量直接从细胞和组织裂解液roteins。此外，通过优化的外周血和排泄物（例如尿）的特定级分的纯化和通过定制和优化某些测定参数，分子型材和低分子浓度的级分（ 例如，外来体的级分）的特征将被检查。

肠易激综合症（IBS）是在胃肠病学中最常见的门诊诊断，困扰10 - 总人口的15％，较健康服务显著负担。对IBS目前，没有普遍公认的诊断标志物（即诊断根据临床症状和排除其他器质性疾患，如胃肠恶性肿瘤，炎性肠疾病和胃肠（GI）感染）。当研究在与亚临床病理学的一般条件，如肠易激综合征，高灵敏度和精确度（再现性）的基因表达谱需要，如在基因表达谱扰动常细微1-3。 miRNA的已被确定为IBS 4,5诊断和功能目标，我们在评估其作为循环IBS-相关的生物扰动3,4,6,7的生物标志物的使用特别感兴趣。 circula的临床应用鼎生物标志物是当前关心的是由于易用性和病人生物标志物&#39;源（如外周血）集合的微创性。 基因表达平台测定法，因为其独特的化学和精简，大多自动化工作流程的，提供一种微阵列平台，提供转录靶向发现的宽和可定制的覆盖范围（在单一反应800目标）。他们还产生高精确度和线性度的表达水平在转录目标量化范围广泛。该测定不需要的RNA的逆转录，得到的cDNA，或任何其它酶反应的扩增。因此，从低丰度或稀有的剪接变异体RNA扩增品种的高循环次数引入潜在的错误是由数字计数的过程中减少。这意味着，各种样品类型，例如纯化总的的RNA（ 即，mRNA的+ miRNA）的，RNA从福尔马林固定的石蜡包埋（FFPE）的标本，以及粗组织和细胞裂解物，可以与测定中使用。 利用一对探针（捕获和报告探针），每个都包含识别关于靶RNA的对应区域的特定靶序列被检测感兴趣的RNA。为了确保短的RNA（ 即，微RNA）的检测，所述成熟miRNA序列通过退火一个独特的寡核苷酸标签（miRtag）到该分子的3&#39;末端延伸。桥接寡核苷酸，其互补于两个成熟靶miRNA和miRNA特异性miRtag的一部分，是用来确保序列特异性。捕获和报告探针分别结扎的3&#39;和5&#39;成熟miRNA-miRtag复杂的结束。捕获探针具有在3&#39;端的生物素标签，这允许它们附加到链霉抗镀膜玻璃载玻片的表面上，FIXI纳克捕获探针的miRNA-miRtag，记者探头复杂的幻灯片。然后电流用来定向在滑动表面上的复合物，使由报告探针的5&#39;端进行荧光条形码使用显微镜和电荷耦合器件（CCD）照相机来可视化。条形码进行计数和解码，产生特定靶miRNA的计数。萤光条形码包含可以通过四个荧光颜色之一占据六个位置，它可以被用来构造4000彩色条形码组合，每编码一个特定的RNA。 样品制备（ 即，RNA纯化或细胞/组织裂解物制剂），以及捕获和报告探针的杂交，以替补进行。十二标本可以在一个幻灯片复用。隔夜杂交后，所有进一步的样品制备是通过机器人准备站进行。然后将装载载玻片传送到广告igital分析器用于成像，计数，解码和数据处理。所得数据被导入到附带的软件，在那里它们被进一步具有高度的用户控制的处理。独特的化学，制备简单，自动性质，简单的数据类型（计数），以及测定的总体简化的工作流程便于高精度基因表达的定量，使得它在功能性病症如学习循环miRNA的表达谱和签名的有用工具肠易激综合征。

<table><tbody><tr><td>nCounter Prep-Station</td><td>Nanostring</td><td>N/A</td><td>Essential</td></tr><tr><td>nCounter Digital Analyzer</td><td>Nanostring</td><td>N/A</td><td>Essential</td></tr><tr><td>Spectrophotometer</td><td>NanoDrop</td><td>ND-1000</td><td>Can use suitable equivalent</td></tr><tr><td>Centrifuge</td><td>Any</td><td>N/A</td><td>Can use suitable equivalent</td></tr><tr><td>MiniMicroCentrifuge</td><td>Any</td><td>N/A</td><td>Can use suitable equivalent</td></tr><tr><td>Pipettes (1,000, 100, 20, 10 &amp;#181;l)</td><td>Any</td><td>N/A</td><td>Can use suitable equivalent</td></tr><tr><td>Qiacube</td><td>Qiagen</td><td>9001292</td><td>Can use suitable equivalent</td></tr><tr><td>PAXgene Blood miRNA Kit</td><td>Qiagen</td><td>763134</td><td>Can use suitable equivalent</td></tr><tr><td>PAXgene Blood Tubes</td><td>VWR</td><td>77776-026</td><td>Can use suitable equivalent</td></tr><tr><td>nCounter Human miRNA Assay Kit</td><td>Nanostring</td><td>150625</td><td>Essential</td></tr><tr><td>nCounter Master Kit</td><td>Nanostring</td><td>100050</td><td>Essential</td></tr><tr><td>nSolver</td><td>Nanostring</td><td>N/A</td><td>Essential</td></tr></tbody></table>

perturbations of circulating mirnas in irritable bowel syndrome detected using a multiplexed high-throughput gene expression platform

基因表达平台测定允许在一个单一的反应高达800转录物（mRNA或微RNA）的表达的健壮和高度可再现的定量。 miRNA的检测直接成像和数字计算的标有颜色编码荧光条码探针集（记者探针和捕获探针）miRNA分子计数成绩单。条形码直接杂交成熟已拉长通过连接一个独特的寡核苷酸标签（miRtag）到3&#39;末端的miRNA。不需要逆转录和转录的扩增。报告探针包含使用四种荧光颜色的组合填充六色的位置的序列。四种颜色在六个位置被用来构建基因特异的彩色条形码序列。杂交后处理机器人准备站自动化。杂交，多余的探针被冲走，而三方结构（采集专业后待的miRNA报告探针）被固定到通过生物素标记的捕捉探针的链霉抗涂覆滑动。成像和条形码数量使用数字分析仪完成的。固定化的条形码的miRNA可视化并使用显微镜和照相机成像，以及独特的条形码被解码并计数。数据质量控制（QC），归一化，并分析由附带测定软件定制设计的数据处理和分析软件促进。该试验证明了在宽范围的表达的高线性度，以及高的灵敏度。样本制备和分析制备不涉及酶反应，逆转录或扩增;有几个步骤;并且在很大程度上自动化，减少了研究者的影响，并导致高的一致性和技术可重复性。在这里，我们描述了在肠易激综合症识别循环的miRNA扰动这种技术的应用。

在功能性障碍转录扰动可能是微妙的，并且为了可靠地检测在表达这些细微的变化，基因表达的定量必须精确。基因表达平台的检测系统通过其高度自动化的性质减少噪音的技术，它采用了独特的化学生产高精度的基因表达数据。技术重复示于图1证明内源性（蓝点）的高再现性和病毒的miRNA（橙色点;管家基因是由黄点表示）表达的定量是可能采用这种方法。使用这种方法，病毒（ 图2a）和内源性（ 图2b）的miRNA，显示从预期表达偏差，由健康研究参与者所定义，可确定为在IBS兴趣目标。应当指出的是，目前还不清楚是否病毒的miRNA这里检测到的派生从整合到人基因组或从病毒载量的病毒基因。由于检测只计算单核苷酸特异性成熟miRNA，具有类似人类内源性的miRNAs交叉杂交的可能性不大。然而，这些分子的差别稳态水平感兴趣的生物标志物。该方法的精确度使得待检测低表达目标中的扰动，因为这些转录物的精确检测不是由更丰富的转录淹没的。微妙的扰动也可以可靠地观察到。 图3和4示出一些在IBS和健康对照组相比的IBS亚型循环的miRNA这些扰动的，和图5和6（均来自3适于）示出的两个最显著差异升高的miRNA （ 图5:miRNA的342-3p， 图6:miRNA的150）。在IBS参与者图6<html>是由相关联的软件生成的结果的图形输出的一个例子。 <img alt="图1" src="/files/ftp_upload/54693/54693fig1.jpg" /> 图 1: 从两个技术重复标准化计数技术重复在两个独立的墨盒运行显示出高的线性相关性（r 2 = 0.90，Y = 1.03x - 0.4）以上的归一化的范围（归一顶端100表达的miRNA）的miRNA计数（在X轴LOG2计数和y轴）。散点图包括黄（8个基因）内源性的miRNA人在蓝色（654的miRNA）的数据，橙色（80微RNA）病毒的内源性的miRNA，和管家基因。内源性病毒的miRNA可在早期版本的测定（1.4版用在这里），但不再包含在标准检测，虽然他们是可自定义添加。/54693fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54693/54693fig2.jpg" /> 图 2: 在IBS-便秘（IBS-℃） 对健康对照 病毒和内源人miRNA的归计数扰动的（a）病毒（橙色）和（b）内源人微RNA（蓝色）在本实施例中，这里是显而易见IBS-C的参与者（Y轴）的miRNA计数（LOG2转化）的回归对健康对照组（X轴）的miRNA的计数（LOG2转化）。大多数miRNA的表达非常相似的两个人群，但一些特别是从相关偏离。这些偏差都是罕见的，大量表达目标之间明显的。 <a href="http://ecsource.jove.com/files/ftp_upload/54693/54693fig2large.jpg" target="_blank">请</a>点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54693/54693fig3.jpg" /> 图3:miRNA的明显与一致差异表达IBS健康对照组相比差异表达的肠易激综合征的miRNA所使用的线性判别分析影响大小的方法，它结合了与检查类别内的一致性统计显着性检验显示， <a href="http://ecsource.jove.com/files/ftp_upload/54693/54693fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54693/54693fig4.jpg" /> 图4:miRNA的明显与一致差异表达IBS亚型健康对照组相比的miRNA迪（:腹泻，-C:-D便秘）在IBS亚型fferentially表示。相对于使用线性判别分析影响大小的方法，它结合了与检查类别内的一致性统计学显着性检验健康对照<a href="http://ecsource.jove.com/files/ftp_upload/54693/54693fig4large.jpg" target="_blank">请点击这里查看大图版本这个数字。</a> <img alt="图5" src="/files/ftp_upload/54693/54693fig5.jpg" /> 图 5: 的miRNA-342-3p的差异表达病因不明的慢性腹痛是肠易激综合征的主要症状。箱形图显示了miR-342-3p（在y轴归一化的计数），原因不明的慢性膀胱疼痛相关的miRNA的，被认为是在整个健康对照组相比所有的IBS亚型更高计数存在于流通。条形代表非离群值范围内，该箱ES的四分位数间距和蓝色正方形的中位数计数。从3修改。 <a href="http://ecsource.jove.com/files/ftp_upload/54693/54693fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54693/54693fig6.jpg" /> 图6: 的miRNA-150的表达差异表明IBS参与者显著高于健康对照组相比（在y轴LOG2转化计数）循环的miR-150计数一把小提琴的情节。该曲线表明在类别计数的频率，竖直棒代表第1 次和第3 次四分位数，和红色正方形表示位数计数。从3修改。 <a href="http://ecsource.jove.com/files/ftp_upload/54693/54693fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform at JoVE.com

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

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JoVE Journal

Genetics

8.2K 次观看.  National Institutes of Health, DHHS. We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome. 

视频: Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

科研

生物工程

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

遗传学

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

转移性结直肠癌患者循环微 rna 自定义面板的检测

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

癌症研究

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

免疫与感染

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

用于隔离和测序微RNA并使用开源工具分析微RNA的完整管道

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

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 ...

医学

Fecal (micro) RNA Isolation

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs from fecal samples of animal and human subjects. The aim is to isolate total RNA with high purity and quantity for downstream analyses such as RNA sequencing, RT-PCR, and micro-array. The advantages of this optimized protocol in the miRNA isolation are capabilities of isolating highly purified RNA products with additional washing steps described, increased quantity of RNA obtained with an improved method in the resuspension of sample, and important tips of decontamination. One limitation is the inability to process and purify larger sample of more than 200 mg as these sample sizes would cause a difficulty in the clear formation of the interphase. Consequently, the large sample size may contaminate the aqueous phase to be extracted as described in the protocol with organic matters that affect the quality of RNA isolated in the end. However, RNA isolates from a sample of up to 200 mg are sufficient for most of downstream analyses.

Fecal micro RNA Isolation

This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR.

粪便（微量）RNA分离

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

生物学

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

摘要

探索更多视频

此视频中的章节

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

转移性结直肠癌患者循环微 rna 自定义面板的检测

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

用于隔离和测序微RNA并使用开源工具分析微RNA的完整管道

脑脊液的microRNA分析采用实时定量PCR技术

粪便（微量）RNA分离

基于探针实时荧光定量PCR途径的小分子RNA定量检测

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

摘要

探索更多视频

此视频中的章节

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

转移性结直肠癌患者循环微 rna 自定义面板的检测

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

用于隔离和测序微RNA并使用开源工具分析微RNA的完整管道

脑脊液的microRNA分析采用实时定量PCR技术

粪便（微量）RNA分离

基于探针实时荧光定量PCR途径的小分子RNA定量检测

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心