Name: probe-based real-time pcr approaches for quantitative measurement of micrornas
Uploaded: 2015-04-14T15:00:00.000+00:00
Duration: 10 min 28 s
Description: Watch this Scientific Journal Video about Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs at JoVE.com

所有的作者承认从NHMRC CTC，悉尼，青少年糖尿病研究基金会（JDRF），澳大利亚的大学和丽贝卡·库珀基金会​​，澳大利亚的基础设施支持。这项研究是通过由澳大利亚研究理事会（FT110100254）和JDRF，澳大利亚（CRN201314）赠款，以AAH WW，配花量和MVJ进行全湿实验室实验，ASJ进行了数据分析。 WW写的第一稿。 AAH计划的研究和分析数据。所有作者阅读并同意提交出版的手稿，人物和工作表的最终版本。

总RNA可从血清使用已建立的在我们的实验室30或使用其它市售的试剂盒的协议进行分离。 注意:用于计算反应体积在每个实验的补充交互式电子表格（用5％过量的体积占移液在内）被提供。 1.探针为基础的实时定量PCR使用标准的96孔板平台<ol><li> cDNA合成（逆转录）在血清/血浆RNA样品的miRNA <ol><li>计算并取出10毫微克的RNA每个cDNA合成反应。加无核酸酶水，使为10ng的RNA的终体积为1.67微升（为一个5微升反应）。不断的冰样本。 </li><li>选择miRNA与解冻他们的RT引物。 </li><li>解冻的RT试剂混合物成分:RT缓冲液（10X），的dNTPs（100毫米），RNA酶抑制剂（20U /微升）。不要让地方酶（重组莫洛尼鼠白血病病毒（rMoMuLV））（recombinanat莫洛尼鼠白血病病毒（rMoMuLV）在冰上逆转录酶（50U /微升）。将它存储于-20℃，直到需要或在冷冻块。 </li><li>制备在冰上的RT试剂混合物，如表1A中所描述。它建议以制备至少5％的过量体积，以补偿移液误差。 </li><li>添加2.33微升的RT试剂混合物的每个成0.2毫升PCR管/板。 </li><li>涡RT管坯混合，然后短暂离心10,000 XG为10秒。添加1微升的miRNA特异性RT引物，以它们各自的PCR管或板。添加1.67微升稀释的RNA，以在该板各自的PCR管或孔中。执行冰全部添加。 </li><li>离心反应管或板，在1950×g离心5分钟，在4℃。 </li><li>设置所述miRNA cDNA合成方案在热循环仪具有以下设定条件:16℃，30分钟，42℃，30分钟，85°下进行5分钟，和4℃保持。 </li><li>设置反应体积至10ul。装入反应管或板进入热循环仪。启动RT运行。存储的cDNA（RT）反应在-20℃下，如果实时PCR扩增不立即进行。 </li></ol></li><li>基于探针的实时定量PCR检测的成熟miRNA的<ol><li>解冻所选探针的基于定量PCR测定（20X），用于相应的RT产物。 </li><li>摇晃瓶子混合实时PCR（定量PCR试剂混合物）的正当性的唯一解决方案。 </li><li>制备的qPCR试剂混合物为每个miRNA进行分析。制备的qPCR获得无菌的1.5 ml离心管，用于每个miRNA样品和添加组件到每个管，如表1B中所描述。它建议以制备至少5％的过量体积，以补偿移液误差。 </li><li>添加4.2微升的各定量PCR试剂混合物的一种光学96孔板（或管）的每个孔中。加入0.8微升各cDNA反应的到各个阱（在步骤1.1的每个miRNA的合成）。 </li><li>封板与适当的光学盖。离心板（或管）在1950×g离心5分钟，在4℃下</li><li>打开计算机，96孔板/微流体阵列读取器和最后启动该软件。确保机器连接正确，正确的块，加热盖（快速96孔板）到位。 </li><li>选择实验为96孔快速块（0.1毫升）中，标准曲线，TaqMan试剂和快速模式。设置的qPCR使用下列程序循环条件的实时PCR检测系统上的:95℃20秒，50个循环（95℃1秒，60℃30秒）。 </li><li>设置反应体积至10ul。装入反应管或板到仪器中。按"开始→运行"。该项目将需要大约1小时即可完成。 </li></ol></li></ol> 2.基于探测器的微流控阵列卡（CA次A和B） 注:基于探测器的miRNA面板当属一套两张384以及微卡（阵列卡A和阵列卡B）。每个卡包含干燥的引物和探针长达380的miRNA和控制。 cDNA产物（有或没有预扩增）特定于卡A或卡B被装到相应的阵列，用于实时PCR。 <ol><li>逆转录（RT）的<ol><li>使用此协议为1-1,000纳克总RNA输入。如果输入是1-350毫微克之间，进行预扩增（前置放大器）的步骤。对于输入超过350毫微克，直接在阵列卡装载的cDNA没有前置放大器。 注:对于血清miRNA表达谱，开始与100毫微克的总RNA。对于一个完整的miRNA的配置文件，运行RT-引物，每个样品RT反应（A组和B组）的两个预定义池。 </li><li>冰上解冻以下试剂。 RT引物（10X）:A组和B组，用的dNTP的dTTP（100毫米），RT缓冲液（10倍）， 氯化镁 （25毫米），RNA酶抑制剂（20 U /＆＃181; 1）。不要让冰酶（重组莫洛尼鼠白血病病毒（rMoMuLV））逆转录酶（50U /微升）。它储存在-20°C，直到需要。 </li><li>然后振荡试剂，除酶，然后短暂离心管以10,000rpm离心10秒。 </li><li>结合试剂，如表2A中描述的，分成两管;一管为池A，其他为池B.每根管子将只包含一个预定义的RT引物的游泳池，无论是从池A或B.游泳池，建议至少准备5％的超额量，以弥补移液错误。 </li><li>颠倒混合，然后短暂离心10,000 XG为10秒。等份100ng的每种RNA样品的进新管中，再加入不含核酸酶的水适当体积，以使总量为3微升。 </li><li>分装4.5微升适当的RT试剂混合到各管的。颠倒混合，然后短暂离心10,000 XG为10秒。在冰上孵育5分钟。 </li><li>地方的样品成热循环，并使用以下条件启动RT:40个循环（16℃，2分钟，42℃1分钟，50℃，1秒），85℃下5分钟，保持在4℃ C.商店的cDNA通过使用RT-引物，这些预定义的池生成在-15至-25℃下或立即使用。 </li></ol></li><li>前置放大<ol><li>解冻的前置放大器引冰上预定义池。轻轻涡引物，然后短暂离心它们在10000 XG为10秒。漩涡的TaqMan前置放大器试剂混合物（2X）轻轻敲击，混匀。 </li><li>结合试剂，如表2B中描述的，分成两管;一管为池A，其他为池B. 注:如上所述，A组前级放大器引物会去管A和B组的引物应该去管B.建议至少准备5％的超额量，以弥补移液错误。 </li><li>颠倒混合，然后短暂离心10,000 XG为10秒。分装22.5微升适当的前置放大器试剂混合到新的试管中。等分试样2.5微升cDNA样品（在RT步骤制备）到相应的管构成。 </li><li>颠倒混合，然后短暂离心10,000 XG为10秒。在冰上孵育5分钟。地方的样品成热循环和启动前置放大器周期。循环条件:95℃，10分钟，55℃2分钟，72℃2分钟，12个循环（95℃15秒，60℃4分钟），99.9℃进行10分钟，保持在4℃下。 </li><li>反转预扩增的cDNA混合，然后短暂离心10,000 XG为10秒。加75微升0.1×TE缓冲液（pH 8.0）中预扩增的cDNA（1:4稀释）。反转稀释前置放大器样品混合，然后短暂离心10,000 XG为10秒。商店稀释的预扩增的cDNA在-15至-25℃下进行长达一周，或立即使用。 </li></ol></li><li>加载微流体卡和执行定量PCR <ol><li>保持微流体miRNA的卡之外，至少一个半小时到达室温。解冻稀释的预扩增的cDNA在冰上和反相管随后简短自旋混合。摇晃瓶子混合定量PCR试剂混合物。 </li><li>在新的管中添加450微升的qPCR试剂混合物至9微升稀释的预扩增的cDNA。对于卡A，使用预扩增产物准备使用底漆游泳池A.添加441微升的核酸自由水，以弥补最终体积为900微升。反转管混合，然后短暂离心10,000 XG为10秒。 </li><li>从包装中取出的TLDA卡（一旦它到达室温），并把它放在与铝箔的一面朝下一个干净的地方。加入100微升的PCR反应混合物到各卡上的8填充端口。有2个端口在每个水库（8水库总每张卡片上）。 注:填充口是在反应混合物中加入了较大的孔，而所述排放口的是更小的孔。每个miRNA TLDA卡有8个水库s，各导致48孔中（24个孔中的一列×2列），从而加载所有384个孔中的卡具有相同的PCR反应混合物。确保A组和每个样品池B都在各自的TLDA卡装。将阵列卡在离心机专业微流控阵列卡持有者桶。 </li><li>用于微流体阵列卡，可以使用合适的离心机（如贺利氏Multifuge 3SR，230V离心机）和特定的桶（如"的TaqMan阵列卡"持有人）。每个桶可容纳3张卡（加载/空）。总是确保一个桶的所有3个时隙被占用并且铲斗通过将类似桶（此铲斗还应包含3卡，或者为空或满）在离心机的相对槽平衡。同时将卡在吊桶座，确保了水库8向上投射，反应井面对离心机的外壁。 <ol><li>旋转卡在331×g离心，在室温下放置1分钟。后第一自旋，打开离心机和视觉确保反应混合物已经通过384口井被分配。重复旋转，在相同的设置1更多的时间。从桶中取出卡，并确保反应混合物中的8个水库的水平是一致的。任何不一致留在水库的液体量使该卡不恰当的进一步使用。 </li></ol></li><li>微流体阵列卡需要一个专门的密封件，其具有精密针头组件（滑架），以密封所述阵列的流体分配通道和均匀分布在所有的孔中的反应混合物（总反应体积1微升/孔）。 </li><li>把马车到起始位置，然后插入装卡与箔面的封口机和列队在封口手写笔针。在一个缓慢的，稳定的和均匀的单冲程，推滑架横跨卡直到它到达密封剂的终点。取出密封阵列卡，然后切断水库期从用剪刀卡。 </li><li>确保正确的块，热盖和样品载体安装在微流控阵实时PCR系统/设备。 </li><li>打开电脑，然后将微流体阵列系统，并最终启动该软件。确保机器连接正确。选择实验，阵列卡，标准曲线，TaqMan试剂和标准模式。导入设置文件无论是卡A卡和B的保存文件。 </li><li>将密封的卡在仪器托盘A1孔在左上角和条形码朝向仪器的前部。按"开始→运行"。该项目将需要大约2小时才能完成。 </li></ol></li></ol> 3.基于探测器的纳米流体人类miRNA面板<ol><li>逆转录（RT）的<ol><li>使用此协议为50-200纳克的总RNA输入，但是100毫微克最适合大多数样品。对于一个完整的miRNA表达谱，运行两个predefi每个样品RT反应（池A和池B）的RT-引物斯内德池。 </li><li>解冻在冰上的试剂:的RT引物集（10X），使用的dNTP的dTTP（100毫米），RT缓冲液（10倍）， 氯化镁，（25毫米），RNA酶抑制剂（20U /微升）预定池。不要让酶（重组莫洛尼鼠白血病病毒（rMoMuLV）逆转录酶（50U /微升）在冰上。将其存放在-20°C，直到需要。然后振荡试剂，除酶，并短暂离心​​它们在10000离心10秒。 </li><li>结合试剂，如表3A中描述的，分成两管;一管为池A，其他为池B.每根管子将只包含一个预定义的RT-引物组的游泳池，无论是从池A或B.游泳池，建议至少准备5％过量体积来弥补移液错误。 </li><li>移液器混合，然后短暂离心在10,000rpm离心10秒。等分试样100中的每个RNA样品进新管中的毫微克，然后加入适当体积的无核酸酶水，使总量为3微升。分装4.5微升适当的RT试剂混合到各自管。 </li><li>颠倒混合，然后短暂离心10,000 XG为10秒。在冰上孵育5分钟。将样品成热循环和启动RT程序。循环条件:40个周期（16℃，2分钟，42℃1分钟，50℃，1秒），85℃下5分钟，保持在4℃。利用RT-引物设定在-15至-25℃的这些预定义的池或立即使用商店的cDNA产生。 </li></ol></li><li>前置放大（前置放大器） <ol><li>解冻的前置放大器底漆冰上设置预先定义的池。轻轻涡底漆，然后短暂离心它们在10000 XG为10秒。漩涡的TaqMan前置放大器试剂混合物（2X）混合。 </li><li>结合试剂，如表3B中描述的，分成两管;一管为池A，其他为池B.每根管子将只包含一个预定义的RT-引物池，要么FROM池A或B.游泳池，建议至少准备5％的过量音量进行补偿取样误差。 </li><li>吸管混合，然后短暂离心它们在10000 XG为10秒。分装22.5微升适当的前置放大器试剂混合到新的试管中。等分试样2.5微升cDNA样品的到各管中。颠倒混合，然后短暂离心管在10000 XG为10秒。在冰上孵育5分钟。 </li><li>将样品成热循环和运行前置放大器。设置反应体积为25微升。循环条件:95℃，10分钟，55℃2分钟，72℃2分钟，12个循环（95℃15秒，60℃4分钟），99.9℃进行10分钟，保持在4℃下。 </li><li>反转预扩增的cDNA混合，然后短暂离心10,000 XG为10秒。在新的管中加入4微升预扩增的cDNA，以156微升0.1×TE缓冲液pH 8.0（1:40稀释）。反转稀释前置放大器样品混合，然后简要CEntrifuge以10,000rpm离心10秒。商店稀释和未稀释的预扩增的cDNA在-15至-25℃下进行长达一周，或立即使用。 </li></ol></li><li>装纳流控阵列和表演定量PCR <ol><li>从使用纳流控阵列幻灯片序号的网站31下载相关的板文件（.TPF）。这包含了特定的纳流控阵幻灯片运行信息。 </li><li>解冻稀释的预扩增的cDNA和TaqMan实时定量PCR试剂混合物（如果使用第一次）在冰上。摇晃瓶子混合定量PCR试剂混合物。在新的管中添加22.5微升的qPCR试剂混合到22.5微升稀释的预扩增的cDNA。轻轻旋涡混匀，然后短暂离心10,000 XG为10秒。 </li><li>等分试样5微升各样品分成8个孔（2列，第4行）的纳流控阵列的工作流384孔样品板。确保每个样品池A和池B在相邻的8孔块（见造型玩具E1或补充的Excel工作表的布局）。 <ol><li>每个样品板可含​​有多达八个幻灯片值得样品。然而，该系统为纳流控阵列仅能处理在单次运行4纳流控阵列。如果超过4幻灯片值得样品是在一个样品板被加载，请确保剩余的部分被密封。轴封采用OpenArray样品板密封。 注意:最好是预先切封口成所需要的部分，所以部分可密封/单独启封，以减少水分蒸发。可替代地，该板可以用一个完整的密封器密封，然后装入时的部分可以单独地切出。 </li></ol></li><li>离心机板在490×g离心在4℃下1分钟。在1小时内加载纳流控阵幻灯片。由于允许密封幻灯片有限的时间，请只加载一次一个幻灯片。除去从冷冻的纳流控阵列载玻片，并允许它来室温erature（〜15分钟）。 </li><li>确保正确的块，热盖和样品载体安装了纳流控阵列系统。打开电脑，和实时PCR系统和装载系统。访问相应的软件，并确保机器相连接。从包装中取出装系统消耗品（阵列滑盖，插头和浸渍液）。 </li><li>轻轻拉动浸渍液的注射器松开的柱塞。取下盖子，放置尖上冲空气从一角。将机器内部装载系统提示和删除盖。将PCR系统内的样板。 </li><li>穿上手套。确保它们紧紧贴合，以尽量减少意外标志着滑盖的风险。小心地打开包装的幻灯片。慢慢倾斜滑入手。请勿触摸滑盖的顶端。 </li><li>放置滑动到PCR系统中，在左边的条形码。从样品板用于装载的部分除去封闭剂。使用加载系统软件进入幻灯片条形码，滑位置，样品位置和小费配置。 </li><li>当所有相关的检查完成后，按负荷幻灯片。而PCR系统加载的幻灯片，从幻灯片盖的底部取下透明红胶。当加载完成后，小心地取出，并在90秒内密封的幻灯片。 <ol><li>放置钢板钳内的幻灯片。将滑盖上的幻灯片。钳30秒。确保盖子被定位成使得条形码正确显示。从板钳取下组装。 </li><li>内滑动位置浸没流体注射器，使得尖端压在盖子。慢慢填入滑动与浸没流体，从而确保沿盖子的流体线路。一旦满，用密封塞滑盖，旋转螺丝，直到手柄脱落。 </li><li>去掉在滑动盖的顶部的塑料盖，然后小心地放入实时PCR系统的滑动载体。确保有苏支持度在幻灯片的底部，因为它正在降低，所以它不会突然下降，而不要触摸滑盖的顶端。它是确定在1小时内触摸滑动/ cassette.Initialize的PCR系统的两侧，启动该程序的qPCR。 </li></ol></li><li>在PCR系统软件中选择"OpenArray"。按"查找幻灯片编号"。这将需要几分钟。如果软件找不到板ID，它会要求它必须手动输入。 </li><li>按"确认板块中心"。同样，这将需要几分钟。检查红点的中心内，有在幻灯片上无指纹/标记。加载各自.TPF文件为每个滑动并指定结果文件名和位置。按"开始→运行"。该项目将需要大约2小时才能完成。 </li></ol></li></ol>

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	<li>Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+animal+microRNAs.">The functions of animal microRNAs.</a> Nature. 431, 350-355 (2004).</li><li>Alvarez-Garcia, I., Miska, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+functions+in+animal+development+and+human+disease.">MicroRNA functions in animal development and human disease.</a> Development. 132, 4653-4662 (2005).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116, 281-297 (2004).</li><li>Joglekar, M. V., Wei, C., Hardikar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+estimation+of+multiple+miRNAs+and+mRNAs+from+a+single+cell.">Quantitative estimation of multiple miRNAs and mRNAs from a single cell.</a> Cold Spring Harb Protoc. 2010, pdb prot5478 (2010).</li><li>Joglekar, M. V., Parekh, V. S., Hardikar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet-specific+microRNAs+in+pancreas+development,+regeneration+and+diabetes.">Islet-specific microRNAs in pancreas development, regeneration and diabetes.</a> Indian J Exp Biol. 49, 401-408 (2011).</li><li>Farr, R. J., Joglekar, M. V., Taylor, C. J., Hardikar, A. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+high-throughput+microRNA+expression+profiling+provides+novel+biomarker+assessment+of+clinical+prostate+and+breast+cancer+biopsies.">Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies.</a> Mol Cancer. 5, 24 (2006).</li><li>Zhu, W., Qin, W., Atasoy, U., Sauter, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+in+breast+cancer+and+healthy+subjects.">Circulating microRNAs in breast cancer and healthy subjects.</a> BMC Res Notes. 2, 89 (2009).</li><li>Zhu, H. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs:+understanding+the+limits+for+quantitative+measurement+by+real-time+PCR.">Circulating microRNAs: understanding the limits for quantitative measurement by real-time PCR.</a> J Am Heart Assoc. 3, e000792 (2014).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+and+Prognostic+Implications+of+a+Serum+miRNA+Panel+in+Oesophageal+Squamous+Cell+Carcinoma.">Diagnostic and Prognostic Implications of a Serum miRNA Panel in Oesophageal Squamous Cell Carcinoma.</a> PLoS One. 9, e92292 (2014).</li><li>McDonald, M. K., Capasso, K. E., Ajit, S. 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第二十加工/出版费用由Life Technologies公司，继在接受本书稿出版的。

内的基于探针的定量PCR的协议的关键步骤，以获得精确的和可再现的结果，以确保1）RT-产品的相同体积和浓度在每个qPCR的反应被装载，2）的部件的正确率和卷所需qPCR的反应制备并充分混合，3）的正确和一致的卷被添加到每个qPCR的反应，和4）每个样品和反应混合物的制备和装载在时间尽可能最短量完成时，同时仍注意到上述关键步骤前面提到的。 微流体阵列卡需要合适的离心机和具体桶。每个桶可容纳3张卡（加载/空）。总是确保一个桶的所有3个时隙被占用并且铲斗通过将类似桶（此铲斗还应包含3卡，或者为空或满）在离心机的相对槽平衡。同时将卡在bucke吨夹持器，确保8储层突出向上和反应孔面对离心机的外壁。旋转卡在331×g离心，在室温下放置1分钟。先旋后，打开离心机和视觉确保反应混合物已经通过384口井被分配。重复旋转，在相同的设置1更多的时间。从桶中取出卡，并确保反应混合物中的8个水库的水平是一致的。任何不一致留在水库的液体量使该卡不恰当的进一步使用。 有RT-产物的相同浓度，RNA的相同的输入浓度被加入到每个RT反应。总RNA的浓度使用微volumespectrophotometer测定。所观察到的二百八分之二百六十零比率可低至1.3用于从血浆/血清中分离的RNA;这似乎没有产生作用于下游的qPCR相关过程或数据生成的30。同样地，260/280比率的此测试的RNA样品1.3-1.7之间，观察到的qPCR无异常影响。 当使用低含量的RNA的样品，如来自生物流体，它可以是难以量化处理之前的RNA。我们建议使用合成穗中的RNA分离和逆转录阶段。根据我们的经验， 拟南芥 miRNA的候补（ATH-的miR-159A和ATH-的miR-172A）是优选的过线虫的miRNA（例如CEL-的miR-39或CEL-的miR-54），这在我们的经验可以具有更高同源性比来自A.拟南芥 。使用这样的阶段特异性尖峰在可占的miRNA的数据跨越测定在不同时间的多个样品的正常化。利用RNA固定输入音量cDNA合成反应还建议32,33。 这里所描述的三个探头基础协议的miRNA定量要求不同数量总RNA输入，不同的工作流程和成本。每个工作流的设计，以满足基于miRNA靶的数目和待分析的样本数不同的吞吐量。随着吞吐量（96 RXN 384 RXN 3072 RXN），每个反应的成本的增加超过单位时间内获得的数据的量减少。因为所有这些平台利用TaqMan探针化学，获得的数据的质量可被预期为相似。的TaqMan定量PCR是一种行之有效的方法，用于识别miRNA的血清/血浆样品9,11,27中的丰度。虽然这里讨论的三个平台共享相同的化学，在将反应体积减少导致转录的检测的动态范围的减小（法尔的RJ 等人 ，未发表的数据）。 96孔定量PCR平台是一个较低的吞吐量，但灵敏度高的平台，并在我们看来，"金标准"为所有基于探测的（或染料为基础）PCR平台。然而，这可能不是最经济或有效的平台如几百或几千个样本被检测和用于多个微RNA。微流体（TLDA）和纳流控（OA）平台设计为允许采集较大的数据在更短的时间高通量/内容平台。尽管批量分歧在TLDA卡被观察，这可以通过从同一批次请求TLDA卡被最小化。我们观察到，TLDA平台（ 图3）显示出显著变化中期的17％ -低丰度的miRNA在使用上的TLDA卡不同批次的相同的样品进行测试。因此，我们建议使用相同批号使用TLDA卡的分析。此变化也可能是由于该技术可变性，包括任何可能的加载和移液误差。但是，我们建议排序/请求同批TLDA卡。没有显著一批变化观察到OA平台上。尽管如此，基于TaqMan experimenTAL定量PCR方法提供了易用性来衡量血浆/血清样品中的miRNA的丰度。这里讨论的低，中，高通量的方法提供了用于分析范围使用一种高效的，可重复和干净的（低的背景噪声）化学样品和miRNA的灵活性。

微RNA（miRNA）是〜22个核苷酸的非编码（NC）的RNA，功能基因表达1-3监管。大多数的miRNA在动物中通过序列特异性碱基配对与mRNA起作用，靶向的3&#39;端非编码区，从而导致该基因表达2-4的负调控。这通常通过抑制mRNA翻译或核糖体落客发生。在循环的miRNA已被证明是在研究和临床方面新颖的生物标志物用于多种疾病，如糖 ​​尿病5-7，卵巢8，前列腺9和乳腺癌10,11，乙型肝炎12和其他自体免疫疾病13。研究已进行识别丰富的miRNA在不同细胞或组织中，以及在从人血浆和血清样品，这是更方便和更小侵入性9,11-15循环。 miRNA的定量不同的方法已经ê使用多个平台，如标准的96孔板平台4,12,16-18中，微流体卡平台12,18-23和纳流控阵列平台17,24配合建立。定量实时PCR（qPCR的）提供了使用多个（染料 - 或基于探测器）化学来测量转录物的相对或绝对数量的能力。基于探针的实时PCR化学提供了低背景荧光和高灵敏度的益处，以检测单个转录拷贝。它相对成本效益，使用简单和高度可再现，使得它青睐的方法用于量化和确定miRNA的表达25。基于探针的定量PCR方法一般包括两个步骤:逆转录（RT）和定量PCR 4,26,27。 RT是其中所述茎 - 环RT引物杂交到一个成熟或初级miRNA分子，并转换为互补的（三）的DNA。该cDNA产物的定量。然后进行了使用的miRNA特异性PCR引物26-28。基于探针的定量PCR的原理是基于互补链延伸在真实时间，它涉及到荧光标记的探针的水解的检测。这些探针被设计为含有荧光报告和淬灭是刚拆开，以允许FRET（荧光共振能量转移）。检测从荧光记者（发射器）发射的由猝灭剂分子的接近掩蔽。当Taq聚合酶（POL）从上游引物延伸，并到达一个拨叉（该探针的5&#39;端），所述的Taq聚合酶外切核酸酶活性水解探针，导致从所述淬灭剂的荧光发射的一个物理分解/分离。此版本的荧光发射的单个分子的记录由检测并呈现为从该井/反应的荧光信号的增量增加。在荧光的增加正比于PCR产物的产生量，从而允许精确的曲antification扩增的靶26,28。 与miRNA的量化的需求不断增加，中高通量技术已发展到允许在很短的时间量要处理样本的数量较多。的TaqMan低密度阵列（TLDA）是基于基于探针的定量PCR化学介质通量微流体创新设计，提供增加的miRNA在一个板上分析的数量。 TLDA涉及使用被用于合成cDNA的RT-引物的预定池。这些cDNA然后被纺成自定义384以及微流体卡，以确定使用qPCR 22,26,29多种miRNA的表达。在卡的各孔含有干燥的引物和探针，以扩增特异的miRNA（S），因此，多达384个反应可以在单一TLDA卡26进行处理。 的纳流控阵列是一种用于检测基因转录物的高通量平台<sup&gt; 24使用相同的探针为基础的化学反应。它利用一个专有矩阵提供疏水-亲水相互作用以方便装载33纳升的反应混合物的成3072的通孔上的不锈钢幻灯片24的阵列。本文重点介绍演示了如何这些方法量化的血清/血浆miRNA的执行，并且必须在执行和解释这些数据时需​​要考虑的重要因素。采取的帐户，他们的个人利益和限制将在本文中进行讨论。

<table><tbody><tr><td>96 well-platform</td><td></td><td></td><td></td></tr><tr><td>For Reverse transcription</td><td></td><td></td><td></td></tr><tr><td>TaqMan MicroRNA Assays INV SM&amp;nbsp;</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4427975</td><td>https://www.lifetechnologies.com/au/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/mirna-ncrna-taqman-assays/single-tube-mirna-taqman-assays.html?ICID=search-4427975&lt;br/&gt; The assays comes as a pack of RT primers and PCR primer.</td></tr><tr><td>TaqMan MicroRNA Reverse Transcription Kit</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4366597 or 4366596</td><td>https://www.lifetechnologies.com/order/catalog/product/4366597?ICID=search-4366597 or http://www.lifetechnologies.com/order/catalog/product/4366596?ICID=search-4366596&lt;br/&gt; The TaqMan MicroRNA Reverse Transcription Kit is the same kit used for reverse transcription in all the threeTaqMan platform- 96-well platform, TLDA and OpenArray.</td></tr><tr><td>For qPCR run on a 96-well platform</td><td></td><td></td><td></td></tr><tr><td>2x TaqMan Fast Universal PCR Master Mix, no AmpErase UNG&amp;nbsp;</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4367846</td><td>https://www.lifetechnologies.com/order/catalog/product/4367846?ICID=search-4367846&lt;br/&gt; The 2X TaqMan Fast Universal PCR Master Mix is used for qPCR on&amp;nbsp; the two TaqMan platforms-96-well platform and TLDA.</td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Applied Biosystems</td><td>4311971</td><td>https://www.lifetechnologies.com/order/catalog/product/4311971?ICID=search-product</td></tr><tr><td>MicroAmp Fast Optical 96-Well Reaction Plate, 0.1 ml&amp;nbsp;</td><td>Applied Biosystems</td><td>4346907</td><td>https://www.lifetechnologies.com/order/catalog/product/4346907?ICID=search-product</td></tr><tr><td>Microfluidics platform (TLDA)</td><td></td><td></td><td></td></tr><tr><td>For Reverse transcription</td><td></td><td></td><td></td></tr><tr><td>Refer to 96-well platform TaqMan MicroRNA Reverse Transcription Kit kit (4366597 or 4366596) (as shown above).</td><td></td><td></td><td></td></tr><tr><td>Megaplex RT Primers, Human Pool A v2.1</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4399966</td><td>https://www.lifetechnologies.com/order/catalog/product/4399966?ICID=search-4399966&lt;br/&gt; The Megaplex RT Primers, Human Pool A v2.1 is used for RT on&amp;nbsp; the two TaqMan platforms- TLDA and OpenArray.</td></tr><tr><td>Megaplex RT Primers, Human Pool B v3.0</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4444281</td><td>https://www.lifetechnologies.com/order/catalog/product/4444281?ICID=search-4444281&lt;br/&gt; The Megaplex RT Primers, Human Pool B v3.0 is used for RT on&amp;nbsp; the two TaqMan platforms- TLDA and OpenArray.</td></tr><tr><td>OR</td><td></td><td></td><td>OR</td></tr><tr><td>Megaplex Primer Pools, Human Pools Set v3.0</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4444750</td><td>http://www.lifetechnologies.com/order/catalog/product/4444750?ICID=search-4444750&lt;br/&gt; The Megaplex Primer Pools, Human Pools Set v3.0 is used for RT on&amp;nbsp; the two TaqMan platforms- TLDA and OpenArray.</td></tr><tr><td>For Pre-amplification</td><td></td><td></td><td></td></tr><tr><td>TaqMan PreAmp Master Mix</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4391128 or 4488593</td><td>https://www.lifetechnologies.com/order/catalog/product/4391128?ICID=search-4391128 or http://www.lifetechnologies.com/order/catalog/product/4488593&lt;br/&gt; The TaqMan PreAmp Master Mix is used for pre-amplification of qPCR on&amp;nbsp; the two TaqMan platforms- TLDA and OpenArray.</td></tr><tr><td>Megaplex PreAmp Primers, Human Pool B v3.0&amp;nbsp;</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4444303</td><td>https://www.lifetechnologies.com/order/catalog/product/4444303?ICID=search-4444303&lt;br/&gt; The Megaplex PreAmp Primers, Human Pool B v3.0 is used for pre-amplification of qPCR on&amp;nbsp; the two TaqMan platforms- TLDA and OpenArray.</td></tr><tr><td>Megaplex PreAmp Primers, Human Pool A v2.1</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4399233</td><td>https://www.lifetechnologies.com/order/catalog/product/4399233?ICID=search-4399233&lt;br/&gt; The Megaplex PreAmp Primers, Human Pool A v2.1 is used for pre-amplification of qPCR on&amp;nbsp; the two TaqMan platforms- TLDA and OpenArray.</td></tr><tr><td>1x TE Buffer (100 ml)</td><td>Invitrogen</td><td>12090-015</td><td>https://www.lifetechnologies.com/order/catalog/product/12090015?ICID=search-product&lt;br/&gt; The 1x TE Buffer is used for pre-amplification of qPCR on&amp;nbsp; the two TaqMan platforms- TLDA and OpenArray.</td></tr><tr><td>To load and run the 384 well microfluidics (TLDA) card</td><td></td><td></td><td></td></tr><tr><td>TaqMan Array Human MicroRNA A+B Cards Set v3.0&amp;nbsp;</td><td>Applied Biosystems</td><td>4444913</td><td>https://www.lifetechnologies.com/order/catalog/product/4444913</td></tr><tr><td>Nanofluidics platform (OpenArray)</td><td></td><td></td><td></td></tr><tr><td>For Reverse transcription</td><td></td><td></td><td></td></tr><tr><td>Refer to 96-well platform TaqMan MicroRNA Reverse Transcription Kit kit (4366597 or 4366596) (as shown above).</td><td></td><td></td><td></td></tr><tr><td>For Pre-amplification</td><td></td><td></td><td></td></tr><tr><td>Refer to TLDA pre-amplification reagents (as shown above)</td><td></td><td></td><td></td></tr><tr><td>To load and run the slides</td><td></td><td></td><td></td></tr><tr><td>TaqMan OpenArray Human MicroRNA Panel, QuantStudio 12K Flex (1 panel)</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4470187</td><td>https://www.lifetechnologies.com/order/catalog/product/4470187?ICID=search-4470187</td></tr><tr><td>QuantStudio 12K Flex OpenArray Accessories Kit&amp;nbsp;</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4469576</td><td>https://www.lifetechnologies.com/order/catalog/product/4469576?ICID=search-4469576</td></tr><tr><td>OpenArray 384-well Sample Plates&amp;nbsp;&amp;nbsp;</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4406947</td><td>https://www.lifetechnologies.com/order/catalog/product/4406947?ICID=search-4406947</td></tr><tr><td>TaqMan OpenArray Real-Time PCR Master Mix</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4462159</td><td>https://www.lifetechnologies.com/order/catalog/product/4462159?ICID=search-4462159</td></tr><tr><td>OpenArray AccuFill System Tips&amp;nbsp;</td><td>Applied Biosystems&lt;br/&gt; &amp;nbsp;</td><td>4457246</td><td>https://www.lifetechnologies.com/order/catalog/product/4457246?ICID=search-4457246</td></tr><tr><td>Others</td><td></td><td></td><td></td></tr><tr><td>Nuclease-Free Water</td><td>Qiagen</td><td>129117</td><td>http://www.qiagen.com/products/catalog/lab-essentials-and-accessories/nuclease-free-water</td></tr></tbody></table>

probe-based real-time pcr approaches for quantitative measurement of micrornas

基于探针的定量PCR（qPCR的）是用于测量转录物丰度的青睐的方法，因为它是，提供了一个准确的和可重复的分析中最灵敏的检测方法之一。基于探针的化学提供了至少背景荧光相对于其他（染料基）化学物质。目前，有可用的使用探针为基础的化学几个平​​台定量转录丰度。定量PCR在96孔板是最经常使用的方法，但是最多只有96个样品或miRNA的可以在单次运行进行测试。这是费时和乏味，如果有大量的样品/ miRNA是待分析。高通量探针为基础的平台，如微流体技术（ 如 TaqMan探针阵列卡）和纳流控阵列（ 例如 OpenArray）提供便于可重复地和有效地检测在短时间内在大量样品的多个微RNA的丰度。在这里，我们展示了实验装置一第二协议用于从血清或血浆-EDTA的样品，使用基于探针的化学和三种不同的平台上（96孔板，微流体和纳米流体阵列）提供的吞吐量水平的不断提高的miRNA定量。

对于miRNA的探针为基础的检测定量PCR反应的推荐量是20微升。注意:我们已经证实，5微升的反应体积是能够产生类似的结果，用20微升体积4,7,30实现。降低反应体积5μl的允许的降低试剂成本的75％，而不在灵敏度明显的损失。如示于图2中 ，反应体积20微升和5微升显示出强大的协同关系，直到39次循环（具有0.92 R 2，p值= 0.0002）。 微流体阵列提供了用于获得对在约5小时（对于卡A和卡B），它是分析多个样品相比于传统的96孔板PCR的更有效的方式表示的样品中的miRNA 754的数据的工具。我们比较了miRNA的微流控芯片阵列卡为同一样品（样品A和样品A重复） 图3A - B显示了一个奥特曼的情节（&lt;STRONG&gt; 3A）及相关的情节（3B）的所有754的miRNA检测这些样品。有随机放置在两个卡（卡A和B）在多个位置3种不同的控制的miRNA（U6，RNU44和RNU48）。当U6循环阈值（CT）值在2运行之间相比，我们没有观察到的值（ 表4）之间的差异显著。同样重要的是要在这里指出，U6是表示在评估样品中更高的丰度（低级Ct值）。然后，我们比较了在两个运行（N = 150）0-19.99之间的Ct值的所有的miRNA，其中有miRNA的类似表达整体与助有效测定的98％（图3C-D）。微RNA与betwee显著差异的数量。 -所有的277的miRNA具有同时在运行20和29.99之间的Ct值，16 miR的原始和重复运行（F图3E）之间显著差异n个运行增加（327 89）30-40时之间被选定为运行（图3G - 高）的Ct值。 的纳流控阵列平台提供的数据从每个血清/血浆样品754的miRNA进行测试，如在图4A中表示的是要研究这些扩增曲线是重要的-因为它是与所有的qPCR -以确保该结果表示真扩增。每个子阵列48（ 图1）还包含一个试验的三个最流行 ​​的"看家"非编码RNA:U6，RNU44和RNU48 图4B示出的U6的典型聚类从单个样本复制。这些重复显示低标准偏差（SD &lt;0.5）等的可靠性的指标。可替代地， 图4C展示的U6重复（SD&gt; 0.5）的第二样品中的增加的可变性。这并没有否定的有效性Øf显示剩余测定中，虽然它确实必要进行更彻底的批评。 U6，与生物体液中最"看家"的miRNA，可具有可变的表达。应当指出的是，样品中，在图4C中所概述的，其中一个将显示4倍以下的U6含量比图4B给出。因为U6在样品中4C呈现的电平为小于75％，开始与在面板4B比1提出，更大的技术可变性是由于泊松分布转录物，这是由小的反应体积17加剧预期。 另一个有用的工具是质量控制（QC）的图像，可用于出口​​，一旦运行完成。精选的这些使用ROX，在定量PCR试 ​​剂混合物中发现的被动染料的荧光，以确认每个通孔已被正确地加载（ 图5）。有通孔，甚或一间<html>轮胎子阵列，可能无法加载由于样品体积不足，蒸发，​​气泡中存在的384孔的样品板，发生故障的Accufill系统或其尖端内完全除去该样品板密封，或缺陷的孔中。任何卸载通孔必须被识别，以避免标签的miRNA为"检测不到"，当在现实中测定从未加载。如果遇到了这个问题，确认至少5微升样品/反应体系的被装入384孔样品板的各孔中，该样品板被正确装载前离心，箔密封件完全除去，并且该加载OpenArray滑动密封并在规定的时间对所有连续运行中运行。如果装载的问题依然存在，这些可能更容易关于特定批次或批号的阵列或相关消耗品和进一步的援助应通过制造商的追捧。 "SRC ="/文件/ ftp_upload / 52586 / 52586fig1.jpg"/&gt; 图1:样本的纳流控阵列工作流程的布局:（A）每384孔样品板可容纳样品多达8纳流控阵列。 （二）稀释，预扩增的cDNA放入8个孔（2列4行），配有游泳池的A和B组中相邻的8孔组。每个圆圈代表一个阱。 （C）的样品板的各孔将被加载到纳流控阵列的一个子阵列。每个小方块代表一个子数组。 <img alt="图2" src="/files/ftp_upload/52586/52586fig2.jpg" /> 图2:合作关系的分析，传统的96孔PCR平台:20微升，并使用标准的96孔板平台CT值（39次）5微升反应体积的TaqMan探针实时定量PCR之间的合作关系。我们比较了4种不同的microRNA（的miR-375中，miR-30C中，miR-30d和的miR-7）在4种不同的人血清和血浆样品。只有11个数据点被绘制，因为其他人无法察觉。 R 2 = 0.92，P = 0.0002。 <img alt="图3" src="/files/ftp_upload/52586/52586fig3.jpg" /> 图3:循环miRNA表达谱使用微流控阵列卡使用2微流体卡（卡A和卡B），754 miRNA的配置文件生成（A - B）。如下所示，我们使用相同的样品进行2微流体阵列运行以检查的微流体卡的结果可重复性。我们观察到miRNA的类似表述的整体，与Ct值0-19.99之间（C - D）。也有少数的miRNAs（277 16）与Ct值（E - F）重复运行之间20-29.99和显著的差异之间。八十九327具有较高的Ct值小分子RNA（30-40）均表现出运行（G - 高）之间的差异显著。 &#160;数据采用配对t检验。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52586/52586fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52586/52586fig4.jpg" /> 图4:PCR产品扩增曲线代表简介:是所有miRNA靶基因的结合（A）扩增曲线的代表人物为人类血浆样品。用于U6的测定（公共控制的ncRNA）被放置在每个子阵列。在（B）的示例演示低变性（SD &lt;0.5），而（C）具有高的标准偏差（SD&gt; 0.5）内U6复制。这两种样品托塔从人血浆中分离升RNA。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52586/52586fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52586/52586fig5.jpg" /> 图5:纳流控阵列的QC分析:一个正确装入纳流控阵列（左）和不正确的加载纳流控阵列（右）的质量控制（QC）的图像。被动染料，ROX（存在于定量PCR试剂混合物），荧光，以指示一个正确装入通孔。在右侧的阵列具有几个子阵列/通孔中未装有定量PCR试剂混合物和这些应该被认定为假阴性PCR反应。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 组件 </td><td> 每5微升反应体积（微升） </td></TR&gt; <tr><td> RT缓冲液（10X） </td><td> 0.5 </td></tr><tr><td>的dNTP（100毫米） </td><td> 0.05 </td></tr><tr><td> RNA酶抑制剂</td><td> 0.6 </td></tr><tr><td>不含核酸酶的水</td><td> 1.39 </td></tr><tr><td>逆转录酶</td><td> 0.33 </td></tr><tr><td>总成交量</td><td> 2.33 </td></tr></tbody></table> 表1A:使用标准的96孔平板平台以5微升RT反应准备的TaqMan实时定量PCR的RT试剂混合物成分。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>组件</td><td>每5微升反应体积（微升） </td><td>每20微升反应体积（微升） </td></tr><tr><td>快速PCR mastermix（2个） </td><td> 2.5 </td><td> 10 </td></tr><tr><td> TaqMan探针定量PCR检测（20倍）* </td><td> 0.25 </td><td> 1 </td></tr><tr><td>无核酸水</td><td> 1.45 </td><td> 5.8 </td></tr><tr><td>总体积: </td><td> 4.2 </td><td> 16.8 </td></tr></tbody></table> *基于所选择的miRNA的测试法的组成部分。 表1B:使用标准的96孔板平台在一个5或20微升的qPCR反应准备的TaqMan实时定量PCR定量PCR试 ​​剂混合物成分。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 组件 </td><td> 每个反应体积（微升） </td></tr><tr><td> Megaplex大楼RT引物（10倍） </td><td> 0.8 </td></tr><tr><td>用的dNTP DTTP（100毫米） </td><td> 0.2 </td></tr><tr><td> Multiscribe逆转录酶（50U /微升） </td><td> 1.5 </td></tr><tr><td> 10X RT缓冲</td><td> 0.8 </td></tr><tr><td>的MgCl 2（25毫摩尔） </td><td> 0.9 </td></tr><tr><td> RNA酶抑制剂＃<html> 160; （20U /微升） </td><td> 0.1 </td></tr><tr><td>无核酸酶水</td><td> 0.2 </td></tr><tr><td>总</td><td> 4.5 </td></tr></tbody></table> 表2A:在一个5微升RT反应准备TLDA miRNA的面板的RT试剂混合物成分。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 组件 </td><td> 每个反应体积（微升） </td></tr><tr><td>荧光定量前置放大器Mastermix（2个） </td><td> 12.5 </td></tr><tr><td> Megaplex大楼前置放大器引物（10倍） </td><td> 2.5 </td></tr><tr><td>不含核酸酶的水</td><td> 7.5 </td></tr><tr><td>总</td><td> 22.5 </td></tr></tbody></table> 表2B:在25μl的预扩增反应准备TLDA miRNA的面板前置放大器试剂混合物成分。 "CELLSPACING ="0"&gt; <tbody><tr><td> 组件 </td><td> 每个反应体积（微升） </td></tr><tr><td> Megaplex大楼RT引物（10倍） </td><td> 0.75 </td></tr><tr><td>用的dNTP DTTP（100毫米） </td><td> 0.15 </td></tr><tr><td> Multiscribe逆转录酶（50U /微升） </td><td> 1.5 </td></tr><tr><td> 10X RT缓冲</td><td> 0.75 </td></tr><tr><td>的MgCl 2（25毫摩尔） </td><td> 0.9 </td></tr><tr><td> RNA酶抑制剂（20 U /微升） </td><td> 0.09 </td></tr><tr><td>无核酸酶水</td><td> 0.35 </td></tr><tr><td>总</td><td> 4.5 </td></tr></tbody></table> 表3A:在5微升RT反应准备探针为基础的纳米流体的miRNA面板RT试剂组合的组件。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 部件s </td><td> 每个反应体积（微升） </td></tr><tr><td>荧光定量前置放大器Mastermix（2个） </td><td> 12.5 </td></tr><tr><td> Megaplex大楼前置放大器引物（10倍） </td><td> 2.5 </td></tr><tr><td>不含核酸酶的水</td><td> 7.5 </td></tr><tr><td>总</td><td> 22.5 </td></tr></tbody></table> 表3B:在25μl的预扩增反应准备探针为基础的纳米流体的miRNA面板前置放大器试剂混合组件注:交互式补充擅长提供了三个平台的电子表格，已经占了5％除了补偿取样误差。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 样品A </td><td> 样品A重复 </td></tr><tr><td> 15.535 </td><td> 16.156 </td></tr><tr><td> 15.471 </td><td> 15.652 </td></tr><tr><td> 15。623 </td><td> 16.063 </td></tr><tr><td> 15.963 </td><td> 15.889 </td></tr><tr><td> 14.006 </td><td> 13.993 </td></tr><tr><td> 14.502 </td><td> 14.623 </td></tr><tr><td> 14.907 </td><td> 14.384 </td></tr><tr><td> 13.732 </td><td> 14.946 </td></tr></tbody></table> 表4:循环利用微流体阵列卡的miRNA U6分析。使用两个微流控阵列卡（A和B）。从样品A的Ct值的U6控制的miRNA（总= 8）。一致U6控制miRNA的丰度之间的运行观察。

Watch this Scientific Journal Video about Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs at JoVE.com

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

循环的microRNA最近出现的前途和新的生物标志物对各种癌症和其他疾病。本文的目的是讨论三种不同的基于探针的实时PCR平台和可用来量化和确定循环微RNA的丰度的方法。

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 ...

probe-based-real-time-pcr-approaches-for-quantitative-measurement

Universidad San Sebastian

Research

JoVE Journal

Biology

33.1K 次观看.  The University of Sydney. 循环的microRNA最近出现的前途和新的生物标志物对各种癌症和其他疾病。本文的目的是讨论三种不同的基于探针的实时PCR平台和可用来量化和确定循环微RNA的丰度的方法。 

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

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

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 ...

医学

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

遗传学

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 ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

生物化学

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 ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

生物学

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

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

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

小RNA分子的高效结扎的微小RNA定量高通量测序

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

基于探针实时荧光定量PCR途径的小分子RNA定量检测

摘要

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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

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

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

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

脑脊液的microRNA分析采用实时定量PCR技术

在乳腺癌细胞雌激素调节小分子RNA的剖析

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

小RNA分子的高效结扎的微小RNA定量高通量测序

基于探针实时荧光定量PCR途径的小分子RNA定量检测

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

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

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

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

脑脊液的microRNA分析采用实时定量PCR技术

在乳腺癌细胞雌激素调节小分子RNA的剖析

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

小RNA分子的高效结扎的微小RNA定量高通量测序

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