这项工作是由CBER / FDA-NIAID / NIH间协议YI-AI-6153-01，FDA / CBER壁间的资金，以及FDA医疗对策倡议的支持。 TCT，MNB，VPM，LMS和KDK被任命为研究参与计划的中心生物制品评价和研究，通过美国能源Departmen和美国之间的间协议给予了美国橡树岭研究所的科学教育支持食品和药物管理局。

1.制备标准系列稀释（图3A） 注：串行稀释系列包含由一个公关/铅一套有针对性的序列线性质粒作为定量标准，定量RT-PCR检测。对于IFN亚型每个标准系列稀释组包含足够的量来运行90检测板。被选择的四个点用于测定中的标准稀释曲线为从20〜32（ 表3）覆盖的Ct值。 <ol><li>解冻，涡旋，并短暂离心​​标准（50分）和鲑鱼精子DNA（单链DNA，10毫克/毫升）储备溶液。 </li><li>通过混合51微升的单链DNA的20.3毫升的水准备的单链DNA /水混合的17个标准稀释套。 </li><li>标签一个8管PCR带的标准稀释集。 注：从库存解决方案的标准系列稀释的制备需要2-3小时。 </li><li>免除190微升的单链DNA /水混合到F在带IRST管和180微升的单链DNA /水搭配，其余五管。 </li><li>通过将10微升从50日下午的标准股票与190微升的单链DNA /水的管道，混合进行1:20稀释;涡并迅速离心PCR带。 </li><li>通过从最近稀释管到该系列中的下一个管输送20微升进行1:10稀释;涡并迅速离心PCR带。 </li><li>重复步骤1.6，直到在稀释系列中的最后管已接收到的标准。 </li><li>每个标准重复步骤1.3-1.7。 </li></ol>见表3所示 2.准备底漆/探针（PR / Pb计）和无模板控制（NTC）工作股票混合料（图3B） 注：每个1.7毫升镨/铅设置工作的股票和128.6微升无模板对照（NTC）的工作股票组合将会使30检测板。 <ol><li>准备镨/铅设置工作的股票混合。 不E：镨/铅套的制备与NTC工作股价从股票混合的解决方案将需要2-3小时。 <ol><li>悬浮用超纯水的引物和探针在100μM的用于制备储备溶液。 </li><li>标签多达17 2ml管;一个用于每个镨/铅集包括在测定中。 </li><li>解冻，涡旋，并短暂离心​​所述引物（100μM），探针（100μM），和单链DNA（10毫克/毫升）储备溶液。 </li><li>加入2微升的单链DNA的使用12.5微升电子多道移液器每管。添加适当的正向引物，反向引物，探针和每个镨/铅设置工作股价管。 见表1靶向人IFN亚型和表2为恒河猴镨/铅套镨/铅套。加水，使每个镨的音量/铅设置工作股价管200微升。 注：引物，探针的体积，并用水补充取决于镨/铅的要求的最终反应浓度设置。 </li></ol></li><li>准备工作NTC股票混合。 <ol><li>标签两节5号管PCR条为NTC工作股混合。 </li><li>转让14.3微升每个镨/铅设置为相应的工作NTC股票混合管。 见表4用于Pr /铅套组合NTC井。加水到各NTC工作原液的混合，使终体积为128.6微升。 </li><li>涡，短暂离心，并放置在黑暗中的管在冰上或在-20℃下长期贮存。 </li></ol></li></ol>见下表4 <ol start="3"><li>淡化镨/铅设置工作的股票。 <ol><li>以下的去除所需的NTC工作原液混合镨/铅套的等分试样，加1.5毫升水，使每个镨/铅的最终体积设定的工作的库存管1.7毫升</li><li>涡，短暂离心，并放置在黑暗中的管在冰上或在-20℃下长期储存。 </li></ol></li></ol><p类=“jove_title”&gt; 3。使用自动化多通道移液器的384孔测定板的准备<ol><li>制备的镨/铅套和NTC混合96孔源板等分至384孔测定板。 注：从镨/铅运作的股票混合源板的制作将需要不到1小时。每个96孔的源板就足以使6 384孔测定板（ 图3C）。 <ol><li>涡和短暂离心镨/铅设置工作股票和NTC工作股价混合。 </li><li>将新的96孔平板在冷藏96-冷却块，并指定将孔为每个镨/铅套或NTC混合各孔接收（参见图2）。 </li><li>用250微升的电子多道移液器加水至96孔板：取66微升的水，以每镨/铅集水井（除了4个孔为目标17）。免除82.5微升水至4目标17口井。免除27.5微升的水，每一个NTC拌匀。 </LI&gt; <li>添加正确镨/铅设定的工作的库存到96孔板的孔中指定：取54微升的正确镨/铅的设置工作原液到指定镨/铅集水井（除了4个孔为目标17）。免除67.5微升目标17镨/铅设置工作股价到指定的目标17镨/铅集水井。添加22.5微升正确的NTC工作的股票组合到指定的井。 </li><li>密封96孔板用粘性板密封并离心1分钟，于700×g离心以确保的内容是在井的底部。 </li><li>使用96孔板适配器放置在96孔板在涡流混合器并混合1分钟，以1,000rpm。离心机的96孔板5分钟，于700×g下。 </li><li>如果使用在同一天存储在黑暗中96孔源板在4℃下，以其他方式储存在-20℃直到使用。 </li></ol></li><li>通过添加镨/铅套使用自动化多通道移液器制备384-孔测定板。 注意：从96孔源板6测定板的制备将采取3-4小时。 <ol><li>之前制作板，预先冷却该冷却巢至4℃的预加热板式蒸发器至125℃，并在底部加热块到80℃用气流20-25升每分钟（LPM）之间吹。 </li><li>开关上的自动化多通道移液器，并打开协议用于使干扰素测定板通过双击软件图标。 </li><li>要建立平台，将在平台位置1完全充满枪头盒，96孔源板位置4，和一个新的384孔板位置6.按下播放按钮开始运行。 注：在运行完成后，每口井将包含5微升了Pr /铅组合。 注：每384孔测定板的孔中加入的ssDNA的最终量为0.025微克。 </li><li>轻轻拍打在平坦表面上的384孔测定板，以确保流体在井的底部，并施加粘合剂板密封。 </li><li>离心机的板在700×g下1分钟。除去粘性板密封，放置384孔测定板进入板机和直接定位384-孔歧管上方的孔中。 </li><li>当在384孔测定板中的内容干燥，应用新的粘性板密封，包裹在铝箔，标记，并存储在黑暗中在4℃下直到使用。 注：将板可以储存在4℃下至少6个月。 </li><li>重复步骤3.2.3-3.2.6直到6×384孔测定板的制备，或在96孔的源板的液体被耗尽。 </li><li>关闭冷却窝，关闭软件，并关闭自动多道移液器。关闭平板机。 </li></ol></li></ol> 4.加载和运行384孔分析板<ol><li>准备两个持家基因（HKG）以及混合物，其由镨/铅集为一个HKG，单链DNA，PCR主混合物，和水的，要添加到干燥的384孔测定板（ <strong&gt;的图3D）。 注：混合料的制备和加载试验板将需要1-2小时。运行试验板会小于2小时。 <ol><li>标签1.5毫升管每个HKG组合。 </li><li>稀2微升的ssDNA（10毫克/毫升）与84.9微升水。添加2微升的稀释的ssDNA，11.8微升水，23微升主混合物，和9.2微升的20×正确HKG镨/镨设置到每个管中，涡旋，并短暂离心​​。 </li><li>使用甘油醛-3-磷酸脱氢酶（GAPDH）和泛素C（UBC）作为HKG为人类测定（见材料表 ）。使用GAPDH和18S核糖体RNA作为HKG为恒河猴测定。 注意：用于执行测定的HKG是柔性的，并应反映适当的细胞类型的基因被测试的GAPDH，卑诗大学，和 18S是常用HKG的例子的模式其他HKG可能更合适。 </li></ol></li><li>准备样品并POSITI已经控制井混合物，其由样品或阳性对照的cDNA，主混合物，和水的，要添加到干燥的384孔测定板。制备样品并在足够数量的阳性对照混合物分配至21孔平板（ 图3D）。 <ol><li>在cDNA合成之前，按照制造商的说明来制备RNA样品用DNA酶消化步骤。 </li><li>预先准备由cDNA合成为至少500纳克的RNA，然后用RNA酶H（24微升各样品最终体积）处理的样品和阳性对照按照制造商的说明进行操作。存储所述制备的cDNA在-20℃直到使用。解冻，旋涡，并简要离心样品的cDNA。 </li><li>添加78.8微升预混和54.8微升的水，每24微升样品和阳性对照管。 </li><li>涡，短暂离心管。 </li></ol></li><li>制备标准和NTC充分混合，CON主混合物和水的sisting，要加入到干燥的384孔测定板（ 图3D）。对于标准的充分混合，在1.5毫升管165微升的水加入到275μl反应混合液。对于NTC充分混合，在1.5毫升管增加52.5微升水52.5μl反应混合液。涡，短暂离心管。 </li><li>制备干燥384-孔测定板装载的混合物和样品。 <ol><li>对于在700×g下1分钟除去干燥384-孔测定板从4°C和离心机。 </li><li>放置板冷冻384-冷却块上，取出粘性板密封，并概述了板的孔来指定，其中的各种混合物和样品将被吸移（ 图1）。 </li></ol></li><li>装载384孔分析板的标准以及搭配，标准，NTC充分混合，样品，阳性对照，并搭配HKG（ 图3E）。分发到板前涡和短暂离心所有解决方案。 <ol><li>免除6微升的标准，充分混合，以每个标准以及与30微升的电子多道移液器。免除1.5微升正确的标准串行稀释到指定好与12.5微升电子多道移液器。 </li><li>免除7.5微升NTC的充分混合，以每NTC很好地与30微升的电子多道移液器。分配7.5微升样品与30微升的电子多道移液器指定孔中。免除2.5微升的HKG镨/铅混合与12.5微升电子多道移液器指定的水井。 </li><li>填写任何空井7.5微升剩水和预混混合物的30微升电子多道移液器的; 7.5微升水仅也会工作。 </li><li>密封384孔测定板与光学粘接膜和离心机在700×g下1分钟。旋涡振荡密封的384孔板在涡流混合器进行2分钟，在2600转离心5分钟，在700×g下。 </li><li>将密封的384孔测定板中的QRT-PCR机中，打开测定布局的模板，并开始运行。 注：可以将结果导出为电子表格或作为分析的文本文件。 </li><li>使用下面的最佳热循环的反应条件的测定：ⅰ）50℃2分钟，ⅱ）95℃，10分钟，ⅲ）95℃25秒，ⅳ）59℃，1分钟。 40个循环重复步骤III和IV。 </li><li>从定量RT-PCR平台的原始数据导出到电子表格应用软件。积设定为标准曲线，观察各线性4点的标准。通过计算ΔCq或通过基于这四个点的标准曲线27的拷贝数进行分析。 </li></ol></li></ol>参见图3所示

<ol>
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Q., 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=Increased+IFN-alpha-Producing+Plasmacytoid+Dendritic+Cells+(pDCs)+in+Human+Th1-Mediated+Type+1+Diabetes:+pDCs+Augment+Th1+Responses+through+IFN-alpha+Production.">Increased IFN-alpha-Producing Plasmacytoid Dendritic Cells (pDCs) in Human Th1-Mediated Type 1 Diabetes: pDCs Augment Th1 Responses through IFN-alpha Production.</a> Journal of immunology. 193, 1024-1034 (2014).</li><li>Hervas-Stubbs, S., 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=Conventional+but+not+plasmacytoid+dendritic+cells+foster+the+systemic+virus-induced+type+I+IFN+response+needed+for+efficient+CD8+T+cell+priming.">Conventional but not plasmacytoid dendritic cells foster the systemic virus-induced type I IFN response needed for efficient CD8 T cell priming.</a> Journal of immunology. 193, 1151-1161 (2014).</li><li>Manry, J., 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=Evolutionary+genetic+dissection+of+human+interferons.">Evolutionary genetic dissection of human interferons.</a> The Journal of experimental medicine. 208, 2747-2759 (2011).</li><li>Chen, J., Baig, E., Fish, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+relatedness+among+the+type+I+interferons.">Diversity and relatedness among the type I interferons.</a> Journal of interferon &amp; cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 24, 687-698 (2004).</li><li>Fox, B. A., Sheppard, P. O., O'Hara, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+genomic+data+in+the+discovery,+annotation+and+evolutionary+interpretation+of+the+interferon-lambda+family.">The role of genomic data in the discovery, annotation and evolutionary interpretation of the interferon-lambda family.</a> PloS one. 4, e4933 (2009).</li><li>Hillyer, P., 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=Expression+profiles+of+human+interferon-alpha+and+interferon-lambda+subtypes+are+ligand-+and+cell-dependent.">Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent.</a> Immunology and cell biology. 90, 774-783 (2012).</li><li>Schramm, L. M., 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=High-throughput+quantitative+real-time+polymerase+chain+reaction+array+for+absolute+and+relative+quantification+of+rhesus+macaque+types+I,+II,+and+III+interferon+and+their+subtypes.">High-throughput quantitative real-time polymerase chain reaction array for absolute and relative quantification of rhesus macaque types I, II, and III interferon and their subtypes.</a> Journal of interferon &amp; cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 32, 407-415 (2012).</li><li>Wang, Q., Chen, L., Long, Y., Tian, H., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+beacons+of+xeno-nucleic+acid+for+detecting+nucleic+acid.">Molecular beacons of xeno-nucleic acid for detecting nucleic acid.</a> Theranostics. 3, 395-408 (2013).</li><li>Little, S., 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=Amplification-refractory+mutation+system+(ARMS)+analysis+of+point+mutations.">Amplification-refractory mutation system (ARMS) analysis of point mutations.</a> Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.]. 9 (Unit 9 8), (2001).</li></ol>

VPM and RLR are co-inventors of the technology to measure expression of IFN- and IFN- subtypes and derive royalty income from it. The remaining authors declare no conflict of interest.

这份报告介绍了设计，批量生产，以及对一个实验来衡量一个研究实验室设置了一组高度相似的基因的转录分析的方法。这里报道的高通量定量RT-PCR测定测量IFN- 和- λ亚型具有高特异性。该方法包括两个主要方面，对同源基因家族的成员和一个生产平台的发展，为创造预装镨/铅套可靠和一致的384孔检测板之间的区分镨/铅套设计。的定量RT-PCR探针结合的结构（MB）或化学（LNA），朝提高其特异性29的方法。为2的引物组，在恒河猴测定（ 表2）中，扩增阻滞突变系统（ARMS）被掺入引物的序列，以进一步提高特异性30。虽然一般来说，最好针对外显子 - 外显子路口ENHA一个QRT-PCR反应的NCE特异性，这是不可能的，因为I型IFN基因缺乏内含子。因此，基因组DNA将在PCR反应中被扩增，并且必须由DNA酶处理RNA提取后降解。 用于Pr /铅套的目标区域被限制在成熟肽的每个干扰素的编码区。由于IFN-α亚型，特别是成熟肽区域之间的高序列相似性，这是有时需要妥协的敏感性，以确保特异性。这是特别用IFN-α17，其中当针对其他的IFN-α亚型相比成熟肽转录只有四个独特碱基的情况。靶向的IFN-α17需要结合多个亚型的转录的引物，限制特异性探针。其结果是，在PCR反应将扩增亚型以外的IFN-α17，从而消耗的PCR试剂和loweri的一个相当大的比例纳克来自特定探针的IFN-α17的荧光信号的幅度。朝向为高度相似的基因，如干扰素设计敏感和特异镨/铅套一个额外的挑战是的可能性，即在GenBank数据库中注释的序列可以是不完整的或全面的在设计的时间。再次，这是对于IFN-α17是一个挑战，在这种新注释的序列不完全一致的序列的数据库中，在设计时的版本。因此，在设计时，镨/铅设置为尚未深入研究基因，这是明智的定期检查靶基因的最新注解序列。最后，这是必要的，以确保镨/铅集不扩增可被转录但不被翻译的假基因。 设计镨/铅套后，接下来的挑战是优化的17个不同镨/铅的PCR条件设置上的一个384孔板。成绩单标准是祁门功夫，功夫坦在测试镨/铅集的特殊性，成为协调众多不同的PCR反应的定量RT-PCR条件是必不可少的。测试镨/铅盘对阵成绩单标准制定的效率和灵敏度;表达高度类似的假基因的质粒可能是必要的，以确保镨/铅设置可选择性地测量的功能性基因的转录。转录标准也提供分析的定量装置（转录物数目），除了相对于管家基因（ΔCq）的半定量分析。 从96孔源板分成多个384孔测定板的机器人移液多信道提高了精度和板间结果的一致性。鲑鱼精子DNA（单链DNA）被用作载体，可以稳定并保留镨/铅套长期贮存，象晒分配到板中的试剂。干燥该板也降低了反应的体积必要可重复的结果，这反过来又保留珍贵样品，并降低了使用昂贵的试剂。通过这些步骤，板批量组装提供精度和一致性超过六个月。 以下板制备，质量控制措施是至关重要的检查批次板的一致性。为了这个目的，一个附加的四组的标准，是一个盘运行。在“5倍标准”板块跟踪性能，并创建一个数据集到一个单独板的标准进行比较。同时测定法设计过程中使用的每个标准的10的10倍稀释液，空间考虑要求四个点被用于在每个测定板的标准曲线，并为5倍的标准板。另外，阳性对照应包括在每个板以使该板的有效性。 通常情况下，需要3-4个小时，从一个镨/铅s准备6检测板环境允许的板;这是准备12板在一个研究实验室的单日可行的。由于每个人的IFN亚型检测板检查17镨/铅套，可容纳15实验样品，一天组装生产了一批板带产生高达3,060实验数据点的能力。从定量RT-PCR技术平台的原始数据可以被处理，并在电子表格应用程序软件用编程脚本自动填充预先设计的分析模板组装。此方法最大程度地减少实际操作的数据输入，从而防止了复制错误，并允许研究者专注于数据分析，而不是数据集。如这里所述，该高通量定量RT-PCR测定可以应用于测量干扰素亚型的人或猕猴样品中的表达，并且可以适用于使用其他种类或同源基因集。板布局的灵活性允许用户改变引物/探针组定制的基因兴趣朝向特定的细胞类型或模型系统。该测定可用于测量IFN表达签名在细胞培养模型学习病原体或疾病模型的上下文患者样品中阐明参与免疫应答的信令机制。

I和III型干扰素（IFN）是在所有脊椎动物1病毒等病原刺激和存在的免疫反应的关键。免疫和非免疫细胞表达和分泌，以及响应于，干扰素2。在检测到相关联的病原体分子模式（PAMP）3,4-先天免疫传感器，如Toll样受体（TLR），刺痛和RIG-I，诱导I型和III干扰素的表达。在人类中，I型干扰素包括IFN-β，-ω，-κ和12亚型IFN-α，并绑定到IFNAR1 / IFNAR2受体复合物2,5。 III型干扰素包括IFN-λ1，-λ2和-λ3并绑定到IL10RB / IL28RA受体复合物2。经典地，I型和III干扰素结合到它们各自的受体复合物，然后招募STAT1 / STAT2异二聚体和引发的干扰素刺激的基因（ISG）6转录。 ISG参与功能的多元化，从抗病毒和抗增殖活性来激活适应性免疫应答7。 病原体已经进化逃避，颠覆，并劫持干扰素信号传导途径的元件的许多机制表明干扰素在先天免疫应答8的重要性。例如，牛痘病毒表达与IFNAR1同源螯合I型IFN 9而雅巴样疾病的病毒分泌的一种糖蛋白，抑制I型和III干扰素蛋白10诱饵受体。除了 ​​其在宿主防御作用，干扰素也牵连在癌症的监视和一些自身炎症性疾病：在乳腺癌细胞中的IFN表达沉默限制免疫监视11，IFN-α的过量生产是在全身的发展机制红斑狼疮12和STING的错误的活化导致造成的STING相关血管病过量IFN的全身炎症反应13。在治疗上，干扰素被用于治疗多发性硬化症14，慢性病毒感染，如HBV 15和HCV 16,17，和癌症，例如多毛细胞白血病18和慢性髓细胞性白血病19。关于干扰素在特定的生理过程的相关问题，连续揭示了该细胞因子家族的普遍存在的性质。 I型干扰素，尤其是IFN-α亚型，通常被认为是作为一个实体20-23，而不是作为一组密切相关的，但明显的，蛋白质。的存在和多种干扰素物种包括IFN-α亚型的持久性，在整个脊椎动物进化24表明，这些亚型中的至少一个子集具有特定或特有的功能。这是可能的干扰素表达的定义的模式将破译并帮助表征1亚型17的一个或多个的特定的功能。研究T的挑战他I型和III干扰素亚型是基于他们共享的序列同一性：所述12的IFN-α亚型共享&gt; 50％25和IFN-λ亚型共享它们的氨基酸序列的81-96％26。在描述的定量RT-PCR检测，分子信标（MB）和锁核酸（LNA）荧光探针区分高度相似的IFN亚型序列之间的单碱基对差异，并允许IFN表达签名的表征。该测定的384-孔板格式包括定量（转录标准）和半定量（持家基因（HKG））的措施，允许通过分别转录拷贝数和ΔCq分析。批量组装，通过干燥该引物/探针（PR / Pb）的促进了自动化多通道移液，和长期存储，可能设置在板上，提高再现性，实用性和实用性测定的。 本协议描述的过程制备384孔的qRT-PCR测定板（ 图1）与靶向人IFN亚型（ 表1）多达17个不同镨/铅套。 PR /铅设定的工作库存源板（ 图2）被用来制备多384孔测定板中，可以使用机器人多道移液器进行自动化的方法。虽然最初的重点是创造一个协议，用于研究人类干扰素表达的签名，这种方法已应用于恒河猴也是如此。虽然该板布局略有不同和镨/铅集（ 表2）是不同的，用于创建人和恒河猴板整体制备方法是相同的。与协议的最小的修改，该方法可以被执行，以允许测定的发展，研究其他组密切相关的基因。 参见图1和2下面 见表1和表2贝尔流

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			<td height="20" style="height:20px;">0.2mL PCR Tube Strips&#160;</td>
			<td>Eppendorf (Westbury, NY, USA)</td>
			<td>E0030 124 286</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">12-channel 384 Equalizer Electronic Pipette, 0.5-12.5uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-139</td>
			<td>*Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-channel 384 Equalizer Electronic Pipette, 1-30uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-137</td>
			<td>*Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-channel Electronic Pipette, 5-250uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td style="width:141px;">14-387-118</td>
			<td>*Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.0mL Micro Centrifuge tube, sterile</td>
			<td>Celltreat Scientific Products (Shirley, MA, USA)</td>
			<td>229446</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-channel Electronic Pipette, 15-1250uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-115</td>
			<td>*Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable Reagent Reservoirs&#160;</td>
			<td>VWR International (Radnor, PA, USA)</td>
			<td>89094-668</td>
			<td>25mL reservoirs with 2 dividers</td>
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		<tr height="20">
			<td height="20" style="height:20px;">E-Centrifuge</td>
			<td>Wealtec Corp. (Sparks, NV, USA)</td>
			<td>1090003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eukaryotic 18S rRNA (18S)</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>Hs99999901_s1</td>
			<td>Rhesus HKG Primer/probe set</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fixed Speed Mini Vortexer</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>02-215-360</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gas Permeable Adhesive Plate Seal</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>AB-0580</td>
			<td>Disposable plate seal used during the plate preparation process&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)</td>
			<td>FBR (Silver Spring, MD, USA)</td>
			<td>Custom Order</td>
			<td>Human HKG Primer/probe set</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>Rh02621745_g1</td>
			<td>Rhesus HKG Primer/probe set</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hudson Solo automated multichannel pipettor&#160;</td>
			<td>Hudson Robotics (Springfield, NJ, USA)</td>
			<td>800200</td>
			<td>Modified with a 384-well cooling nest</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Linearized plasmids (IFN genes)</td>
			<td>Aldevron (Fargo, ND, USA)</td>
			<td>Custom Order</td>
			<td>Item 3000, 3580; used for assay standards&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LNA inhibitors</td>
			<td>Exiqon (Woburn, MA, USA)</td>
			<td>Custom Order</td>
			<td>Item 500100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LNA Probes</td>
			<td>Sigma-Proligo (St. Louis, MO, USA)</td>
			<td>Custom Order</td>
			<td>Material number VC00023</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix Filtered Pipet Tips, 12.5uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-193</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix Filtered Pipet Tips, 30uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-196</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix TallTip Extended Length Pipet Tips, 1250uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix TallTip Extended Length Pipet Tips, 250uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Optical 384-Well Reaction Plate with Barcode</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>4309849</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Optical 96-Well Reaction Plate</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>N8010560</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Optical Adhesive Film</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>4311971</td>
			<td>Adhesive film used for sealing the plate prior to the qRT-PCR run&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsoft Excel 2010 Spreadsheet</td>
			<td>Microsoft (Redmond, WA, USA)&#160;</td>
			<td>Office 2010</td>
			<td>Visual Basic programming language&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MixMate Vortex Mixer</td>
			<td>Eppendorf (Westbury, NY, USA)</td>
			<td>5353 000.014</td>
			<td>2 dimensional plate vortex apparatus</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Beacon Probes</td>
			<td>FBR (Silver Spring, MD, USA)</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR Cooler, iceless cold storage system for 96 well plates and PCR tubes</td>
			<td>Eppendorf (Westbury, NY, USA)</td>
			<td>Z606634-1EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plate Dryer (manifold)</td>
			<td>Mechanical and Electrical Design Fabrications, NIH Office of Research Services (Bethesda, MD, USA)</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primer sets</td>
			<td>FBR (Silver Spring, MD, USA)</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC57 plasmid DNA</td>
			<td>Genescript (Piscataway, NJ, USA)</td>
			<td>SD1176</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rnase-Free 1.5mL Microfuge Tubes</td>
			<td>Life Technologies (Grand Island, NY, USA)</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-free DNase Set (50)</td>
			<td>Qiagen (Valencia, CA, USA)</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase H</td>
			<td>New England Biolabs (Ipswitch, MA, USA)</td>
			<td>M0297L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit (250)</td>
			<td>Qiagen (Valencia, CA, USA)</td>
			<td>74106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Robot Tips (clear tip pre-sterilized pipet tips)</td>
			<td>Hudson Robotics (Springfield, NJ, USA)</td>
			<td>800350-S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Salmon Sperm DNA Solution</td>
			<td>Invitrogen (Carlsbad, CA, USA)</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SoftLinx Version V</td>
			<td>Hudson Robotics (Springfield, NJ, USA)</td>
			<td>Custom Order</td>
			<td>Software for programming pipetting steps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Fast Universal PCR Master Mix (2x), no AmpErase UNG</td>
			<td>Life Technologies (Grand Island, NY, USA)</td>
			<td>4352042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ABgene Adhesive Plate Seals&#160;&#160;</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>AB0580</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">Ubiquitin C (UBC)</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>Hs01871556_s1</td>
			<td>Human HKG Primer/probe set</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Verso cDNA synthesis Kit</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>AB1453B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ViiA 7 Real-Time PCR System</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>4453536</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water-Ultra Pure</td>
			<td>Quality Biological, INC. (Gaithersburg, MD, USA)</td>
			<td>351-029-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zipvap 384 (plate dryer heating element)</td>
			<td>Glas-Col LLC (Terre Haute, IN, USA)</td>
			<td>384-109A</td>
			<td>Plate Dryer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput quantitative real-time rt-pcr assay for determining expression profiles of types i and iii interferon subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

的定量RT-PCR测定描述可以实现以分析的I型和III干扰素的表达模式在多种细胞类型和上下文。例如，人I型和III干扰素表达的签名中的外周血单核细胞（PBMC）从6供体与TLR配体刺激进行分析;聚I：C（25微克/毫升），LPS（10毫微克/毫升），咪喹莫特（10μM），和CpG寡核苷酸（1μM）（ 图4）。使用电子表格应用软件数据进行分析，并作为雷达图表具有根据它们的蛋白质序列27的系统发生树布置顺时针IFN-α亚型。人IFN表达的雷达图表示于使用掺入测定法设计分析的两种不同的方法来计算对数标尺：标准化为HKG（ΔCq）绝对CQ值，和每总RNA微克（微克）复制模板的数。拷贝数值从结果○计算<html>发成绩单的标准曲线。该数据表明，通过TLR激动剂诱发人IFN表达签名是配体特异性。 参见图4所示 这表现为人类干扰素表达的签名，我和III IFN在恒河猴的类型表达签名还具体TLR配体。 PBMC从3个供体刺激用聚I：C（50微克/毫升），LPS（10微克/毫升），和咪喹莫特（10微克/ ml）处理3小时（ 图5）。在未受刺激的细胞在基线IFN亚型表达水平较低。的IFN-α亚型数量有限表达响应LPS和聚I：C。相反，在响应于咪喹莫特的IFN表达高，并且亚型表达宽。 IFN-β和IFN-λ1的表达增强被所有3，TLR激动剂28。 参见图5所示 总是“&gt; <img alt="图1" src="/files/ftp_upload/52650/52650fig1.jpg" /> 图1：一个384孔分析板17镨/铅套 ， 布局目标数是指公关/铅集。四点标准曲线（暗灰色三角形）加入列1-5。所述HKG镨/铅套（白色背景）加入到列6和7的其余列，8-24，特异于的17镨/铅集之一。样品加入到行的AP，从列6-24（黑色箭头）。两口井（O5，P5）在第5栏的底部只接收水和母液。 <a href="https://www.jove.com/files/ftp_upload/52650/52650fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52650/52650fig2.jpg" /> 图2：一个96孔源板17镨/铅套布局的目标数是指公关/铅设置。黑色的箭头表示当镨/铅集添加到多个井。 NTC混合物被添加到指定的孔中（暗灰色背景）。两口井（F3，G3）与斜线未使用。 <a href="https://www.jove.com/files/ftp_upload/52650/52650fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52650/52650fig3.jpg" /> 图3：示意图中的定量RT-PCR检测协议的具体步骤 AE图选择协议的一部分。 （A）第1步：标准系列稀释准备。 （B）步骤2：镨/ Pb和NTC工作的股票混合准备。 （C）步骤3.1：的镨/铅套和NTC混合物在96孔加工股板的制备。 （D）的步骤4.1-3：混合料用于装载384孔测定板的制备。 （美东时间EP 4.5：加载384孔测定板。图被从左到右下角的顶部读出。试剂为干扰素（IFN）测定被储存在-20℃，都列在左边的列;在协议行单独行动。 <a href="https://www.jove.com/files/ftp_upload/52650/52650fig3highres.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/52650/52650fig4.jpg" /> 图4：在外周血单核细胞的人IFN表达签名（PBMC）中的不同响应于用于刺激的PBMC刺激TLR配体和RNA收获用于定量RT-PCR分析的TLR配体。高峰响应的几何平均值与聚I：C（25微克/毫升）在8小时（红色正方形），LPS（10毫微克/毫升）在4小时（绿色三角形），咪喹莫特（10μM）在16小时（紫色菱形），CpG​​的（1μM）在16小时（黑色圆圈），和未刺激的对照，在16小时（蓝色圆圈）从6供体示于日志10刻度作为HKG UBCΔCq（左）的表达的功能或按核糖核酸微克拷贝数（右） 。 IFN-α亚型都根据氨基酸序列相似性的亲缘情节进行排序。这个数字最初发表在免疫学和细胞生物学27。 <img alt="图5" src="/files/ftp_upload/52650/52650fig5.jpg" /> 图5：在外周血单核细胞的猕猴IFN表达签名（PBMC），以响应用于刺激 PBMC从三个恒河猴（由正方形，菱形，三角形和指定） 的TLR配体不同于全血中分离和刺激脂多糖（LPS）（10微克/毫升），聚I：C（50微克/毫升）或咪喹莫特（10微克/毫升）。细胞在0小时收获（OPEñ形状），或3小时的TLR刺激（封闭的形状），干扰素的表达测量后。型I，II和III的IFN转录水平作为HKG GAPDHΔCq（左）的表达的功能，或作为拷贝数/微克的RNA（右）显示在日志10的规模。这个数字最初发表在Journal of干扰素与细胞因子研究28。 <img alt="表1" fo:content-="" fo:content-width="7in" src="/files/ftp_upload/52650/52650table1.jpg" width="700" /> 表1：人类IFN底漆/探针组序列和反应的信息 <a href="https://www.jove.com/files/ftp_upload/52650/52650table1.jpg" target="_blank">请点击这里查看此表的放大版本。</a> <img alt="表2" fo:content-="" fo:content-width="7in" src="/files/ftp_upload/52650/52650table2.jpg" width="700" /> 表2：恒河猴IFN引物/探针组序列和反应的信息 <a href="https://www.jove.com/files/ftp_upload/52650/52650table2.jpg" target="_blank">请点击这里查看此表的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td colspan="4"> 质粒浓度（FM） </td></tr><tr><td></td><td>一</td><td> B </td><td> Ç </td><td> ð </td></tr><tr><td> IFN-α1 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td><td> 0.025 </td></tr><tr><td> IFN-α2 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td><td> 0.025 </td></tr><tr><td> IFN-α4 </td><td> 2500 </td><td> 250 </td><td> 25 </td><td> 2.5 </td></tr><tr><td> IFN-α5 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td><td> 0.025 </td></tr><tr><td> IFN-α6 </td><td> 250 </td><td> 25 </td><td> 2.5 </td><td>0.25 </td></tr><tr><td> IFN-α7 </td><td> 250 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td></tr><tr><td> IFN-α8 </td><td> 250 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td></tr><tr><td> IFN-α10 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td><td> 0.025 </td></tr><tr><td> IFN-α14 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td><td> 0.025 </td></tr><tr><td> IFN-α16 </td><td> 250 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td></tr><tr><td> IFN-α17 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td><td> 0.025 </td></tr><tr><td> IFN-α21 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td><td> 0.025 </td></tr><tr><td> IFN-λ1 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td><td> 0.025 </td></tr><tr><td> IFN-λ2 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td><td> 0.025 </td></tr><tr><td> IFN-λ3 </td><td>250 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td></tr><tr><td> IFN-β </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td><td> 0.025 </td></tr><tr><td> IFN-ω </td><td> 250 </td><td> 25 </td><td> 2.5 </td><td> 0.25 </td></tr></tbody></table> 表3：标准品稀释设定浓度信息。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 加IFN镨/铅套 </td><td> 加水量（毫升） </td></tr><tr><td> NTC 1 </td><td> IFN-β，-ω，-λ3 </td><td> 85.7 </td></tr><tr><td> NTC 2 </td><td> IFN-α1，-α5 </td><td> 100.0 </td></tr><tr><td> NTC 3 </td><td> IFN-α2 </td><td> 114.3 </td></tr><tr><td> NTC 4 </td><td> IFN-α4 </td><td> 114.3 </td></tr><tr><td> NTC 5 </td><td> IFN-α7 </td><td> 114.3 </td></tr><tr><td>NTC 6 </td><td> IFN-α6，-α8，-α10 </td><td> 85.7 </td></tr><tr><td> NTC 7 </td><td> IFN-α14，-α16 </td><td> 100.0 </td></tr><tr><td> NTC 8 </td><td> IFN-α17 </td><td> 114.3 </td></tr><tr><td> NTC 9 </td><td> IFN-α21，-λ1 </td><td> 100.0 </td></tr><tr><td> NTC 10 </td><td> IFN-λ2 </td><td> 114.3 </td></tr></tbody></table> 表4：NTC工作的股票混合的信息。

Watch this Scientific Journal Video about High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes at JoVE.com

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

high-throughput-quantitative-real-time-rt-pcr-assay-for-determining

Research

JoVE Journal

Immunology and Infection

13.2K 次观看.  US Food and Drug Administration. This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible. 

视频: High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

科研

免疫与感染

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and protection to the underlying cells. Analysis of tears is an emerging area for the identification of biomarkers for the prediction, diagnosis, and prognosis of various ocular diseases. Tears are easily accessible and their collection is non-invasive. Therefore, advancing technologies are gaining prominence for identification of multiple analytes in tears to study changes in protein or metabolite composition and its association with pathological conditions. Tear cytokines are ideal biomarkers for studying the health of the ocular surface and also help in understanding the mechanisms of different ocular surface disorders like dry eye disease and vernal conjunctivitis. Bead based multiplex assays have the capability of detecting multiple analytes in a small amount of sample with a higher sensitivity. Here we describe a standardized protocol of tear sample collection, extraction and analysis of cytokine profiling using a bead based multiplex assay.

Analysis of tear film cytokines helps in studying various ocular diseases. Bead based multiplex assays are simple and sensitive and enable the testing of multiple targets in samples with small volumes. Here we describe a protocol for tear film cytokine profiling a using bead based multiplex assay.

基于磁珠复合法的撕裂细胞因子分析

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and ...

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

用三维定量相位成像和机器学习技术对淋巴细胞亚型进行无标签鉴定

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.

用于狂犬病诊断的泛源病毒实时RT-PCR

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

微生物相互作用研究的高通量共培养测定

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of the disease.&#160;To study such processes, researchers can use high-throughput sequencing techniques (RNA-seq) that measure the dynamic changes of the host transcriptome at different stages of infection, clinical outcomes, or disease severity.This investigation can lead to a better understanding of the diseases, as well as uncovering potential drug targets and treatments. The protocol presented here describes a complete pipeline to analyze RNA-sequencing data from raw reads to functional analysis. The pipeline is divided into five steps: (1) quality control of the data; (2) mapping and annotation of genes; (3) statistical analysis to identify differentially expressed genes and co-expressed genes; (4) determination of the molecular degree of the perturbation of samples; and (5) functional analysis. Step 1 removes technical artifacts that may impact the quality of downstream analyses. In step 2, genes are mapped and annotated according to standard library protocols.&#160;The statistical analysis in step 3 identifies genes that are differentially expressed or co-expressed in infected samples, in comparison with non-infected ones. Sample variability and the presence of potential biological outliers are verified using the molecular degree of perturbation approach in step 4. Finally, the functional analysis in step 5 reveals the pathways associated with the disease phenotype. The presented pipeline aims to support researchers through the RNA-seq data analysis from host-pathogen interaction studies and drive future&#160;in vitro or in vivo experiments, that are essential to understand the molecular mechanism of infections.

The protocol presented here describes a complete pipeline to analyze RNA-sequencing transcriptome data from raw reads to functional analysis, including quality control and preprocessing steps to advanced statistical analytical approaches.

用于研究宿主-病原体相互作用的高通量转录组分析

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of ...