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>
	<li>Yan, N., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+antiviral+immunity.">Intrinsic antiviral immunity.</a> Nature. 13, 214-222 (2012).</li><li>Pestka, S., Krause, C. D., Walter, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferons,+interferon-like+cytokines,+and+their+receptors.">Interferons, interferon-like cytokines, and their receptors.</a> Immunological reviews. 202, 8-32 (2004).</li><li>Baum, A., Garcia-Sastre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+type+I+interferon+by+RNA+viruses:+cellular+receptors+and+their+substrates.">Induction of type I interferon by RNA viruses: cellular receptors and their substrates.</a> Amino acids. 38, 1283-1299 (2010).</li><li>Kotenko, S. V., 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=IFN-lambdas+mediate+antiviral+protection+through+a+distinct+class+II+cytokine+receptor+complex.">IFN-lambdas mediate antiviral protection through a distinct class II cytokine receptor complex.</a> Nature. 4, 69-77 (2003).</li><li>Hardy, M. P., Owczarek, C. M., Jermiin, L. S., Ejdeback, M., Hertzog, 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=Characterization+of+the+type+I+interferon+locus+and+identification+of+novel+genes.">Characterization of the type I interferon locus and identification of novel genes.</a> Genomics. 84, 331-345 (2004).</li><li>Cheon, H., 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=IFNbeta-dependent+increases+in+STAT1,+STAT2,+and+IRF9+mediate+resistance+to+viruses+and+DNA+damage.">IFNbeta-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage.</a> The EMBO journal. 32, 2751-2763 (2013).</li><li>Schneider, W. M., Chevillotte, M. D., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-stimulated+genes:+a+complex+web+of+host+defenses.">Interferon-stimulated genes: a complex web of host defenses.</a> Annual review of immunology. 32, 513-545 (2014).</li><li>Hiscott, J., Nguyen, T. L., Arguello, M., Nakhaei, P., Paz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+the+nuclear+factor-kappaB+pathway+and+the+innate+immune+response+by+viruses.">Manipulation of the nuclear factor-kappaB pathway and the innate immune response by viruses.</a> Oncogene. 25, 6844-6867 (2006).</li><li>Alcami, A., Symons, J. A., Smith, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vaccinia+virus+soluble+alpha/beta+interferon+(IFN)+receptor+binds+to+the+cell+surface+and+protects+cells+from+the+antiviral+effects+of+IFN.">The vaccinia virus soluble alpha/beta interferon (IFN) receptor binds to the cell surface and protects cells from the antiviral effects of IFN.</a> Journal of virology. 74, 11230-11239 (2000).</li><li>Huang, 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=Inhibition+of+type+I+and+type+III+interferons+by+a+secreted+glycoprotein+from+Yaba-like+disease+virus.">Inhibition of type I and type III interferons by a secreted glycoprotein from Yaba-like disease virus.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 9822-9827 (2007).</li><li>Bidwell, B. N., 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=Silencing+of+Irf7+pathways+in+breast+cancer+cells+promotes+bone+metastasis+through+immune+escape.">Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape.</a> Nature medicine. 18, 1224-1231 (2012).</li><li>Di Domizio, J., Cao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fueling+autoimmunity:+type+I+interferon+in+autoimmune+diseases.">Fueling autoimmunity: type I interferon in autoimmune diseases.</a> Expert review of clinical immunology. 9, 201-210 (2013).</li><li>Crow, Y. J., Casanova, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STING-Associated+Vasculopathy+with+Onset+in+Infancy+-+A+New+Interferonopathy.">STING-Associated Vasculopathy with Onset in Infancy - A New Interferonopathy.</a> The New England journal of medicine. , (2014).</li><li>Bermel, R. A., Rudick, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-beta+treatment+for+multiple+sclerosis.">Interferon-beta treatment for multiple sclerosis.</a> Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics. 4, 633-646 (2007).</li><li>Kingham, J. G., 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=Treatment+of+HBsAg-positive+chronic+active+hepatitis+with+human+fibroblast+interferon.">Treatment of HBsAg-positive chronic active hepatitis with human fibroblast interferon.</a> Gut. 19, 91-94 (1978).</li><li>Heathcote, J., Main, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+hepatitis+C.">Treatment of hepatitis C.</a> Journal of viral hepatitis. 12, 223-235 (2005).</li><li>Donnelly, R. P., Dickensheets, H., O'Brien, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-lambda+and+therapy+for+chronic+hepatitis+C+virus+infection.">Interferon-lambda and therapy for chronic hepatitis C virus infection.</a> Trends in immunology. 32, 443-450 (2011).</li><li>Castaigne, 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=Interferon+alpha+in+the+treatment+of+hairy+cell+leukemia.">Interferon alpha in the treatment of hairy cell leukemia.</a> Cancer. 57, 1681-1684 (1986).</li><li>Kumar, L., Basade, M., Gulati, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+interferon-alpha+in+chronic+myeloid+leukemia.">Role of interferon-alpha in chronic myeloid leukemia.</a> The Journal of the Association of Physicians of India. 3, 26-32 (1995).</li><li>Dai, 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=Interferon+alpha+on+NZM2328.Lc1R27:+Enhancing+autoimmunity+and+immune+complex-mediated+glomerulonephritis+without+end+stage+renal+failure.">Interferon alpha on NZM2328.Lc1R27: Enhancing autoimmunity and immune complex-mediated glomerulonephritis without end stage renal failure.</a> Clinical immunology (Orlando, Fla.). 154, 66-71 (2014).</li><li>Maeda, 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=Interferon-alpha+Acts+on+the+S/G2/M+Phases+to+Induce+Apoptosis+in+the+G1+Phase+of+an+IFNAR2-Expressing+Hepatocellular+Carcinoma+Cell+Line.">Interferon-alpha Acts on the S/G2/M Phases to Induce Apoptosis in the G1 Phase of an IFNAR2-Expressing Hepatocellular Carcinoma Cell Line.</a> The Journal of biological chemistry. , (2014).</li><li>Xia, C. 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

<table><tbody><tr><td>0.2 ml PCR Tube Strips&amp;nbsp;</td><td>Eppendorf (Westbury, NY, USA)</td><td>E0030 124 286</td><td></td></tr><tr><td>12-channel 384 Equalizer Electronic Pipette, 0.5-12.5 ul</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>14-387-139</td><td>*Discontinued</td></tr><tr><td>12-channel 384 Equalizer Electronic Pipette, 1-30 ul</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>14-387-137</td><td>*Discontinued</td></tr><tr><td>12-channel Electronic Pipette, 5-250 ul</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>14-387-118</td><td>*Discontinued</td></tr><tr><td>2.0 ml Micro Centrifuge tube, sterile</td><td>Celltreat Scientific Products (Shirley, MA, USA)</td><td>229446</td><td></td></tr><tr><td>8-channel Electronic Pipette, 15-1250 ul</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>14-387-115</td><td>*Discontinued</td></tr><tr><td>Disposable Reagent Reservoirs&amp;nbsp;</td><td>VWR International (Radnor, PA, USA)</td><td>89094-668</td><td>25mL reservoirs with 2 dividers</td></tr><tr><td>E-Centrifuge</td><td>Wealtec Corp. (Sparks, NV, USA)</td><td>1090003</td><td></td></tr><tr><td>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><td>Fixed Speed Mini Vortexer</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>02-215-360</td><td></td></tr><tr><td>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&amp;nbsp;</td></tr><tr><td>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><td>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><td>Hudson Solo automated multichannel pipettor&amp;nbsp;</td><td>Hudson Robotics (Springfield, NJ, USA)</td><td>800200</td><td>Modified with a 384-well cooling nest</td></tr><tr><td>Linearized plasmids (IFN genes)</td><td>Aldevron (Fargo, ND, USA)</td><td>Custom Order</td><td>Item 3000, 3580; used for assay standards&amp;nbsp;</td></tr><tr><td>LNA inhibitors</td><td>Exiqon (Woburn, MA, USA)</td><td>Custom Order</td><td>Item 500100</td></tr><tr><td>LNA Probes</td><td>Sigma-Proligo (St. Louis, MO, USA)</td><td>Custom Order</td><td>Material number VC00023</td></tr><tr><td>Matrix Filtered Pipet Tips, 12.5 ul</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>14-387-193</td><td></td></tr><tr><td>Matrix Filtered Pipet Tips, 30 ul</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>14-387-196</td><td></td></tr><tr><td>Matrix TallTip Extended Length Pipet Tips, 1,250 ul</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>14-387-160</td><td></td></tr><tr><td>Matrix TallTip Extended Length Pipet Tips, 250 ul</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>14-387-152</td><td></td></tr><tr><td>MicroAmp Optical 384-Well Reaction Plate with Barcode</td><td>Applied Biosystems (Foster City, CA, USA)</td><td>4309849</td><td></td></tr><tr><td>MicroAmp Optical 96-Well Reaction Plate</td><td>Applied Biosystems (Foster City, CA, USA)</td><td>N8010560</td><td></td></tr><tr><td>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&amp;nbsp;</td></tr><tr><td>Microsoft Excel 2010 Spreadsheet</td><td>Microsoft (Redmond, WA, USA)&amp;nbsp;</td><td>Office 2010</td><td>Visual Basic programming language&amp;nbsp;</td></tr><tr><td>MixMate Vortex Mixer</td><td>Eppendorf (Westbury, NY, USA)</td><td>5353 000.014</td><td>2 dimensional plate vortex apparatus</td></tr><tr><td>Molecular Beacon Probes</td><td>FBR (Silver Spring, MD, USA)</td><td>Custom Order</td><td></td></tr><tr><td>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><td>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><td>Primer sets</td><td>FBR (Silver Spring, MD, USA)</td><td>Custom Order</td><td></td></tr><tr><td>pUC57 plasmid DNA</td><td>Genescript (Piscataway, NJ, USA)</td><td>SD1176</td><td></td></tr><tr><td>Rnase-Free 1.5 ml Microfuge Tubes</td><td>Life Technologies (Grand Island, NY, USA)</td><td>AM12400</td><td></td></tr><tr><td>RNase-free DNase Set (50)</td><td>Qiagen (Valencia, CA, USA)</td><td>79254</td><td></td></tr><tr><td>RNase H</td><td>New England Biolabs (Ipswitch, MA, USA)</td><td>M0297L</td><td></td></tr><tr><td>RNeasy Mini Kit (250)</td><td>Qiagen (Valencia, CA, USA)</td><td>74106</td><td></td></tr><tr><td>Robot Tips (clear tip pre-sterilized pipet tips)</td><td>Hudson Robotics (Springfield, NJ, USA)</td><td>800350-S</td><td></td></tr><tr><td>Salmon Sperm DNA Solution</td><td>Invitrogen (Carlsbad, CA, USA)</td><td>15632-011</td><td></td></tr><tr><td>SoftLinx Version V</td><td>Hudson Robotics (Springfield, NJ, USA)</td><td>Custom Order</td><td>Software for programming pipetting steps</td></tr><tr><td>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><td>ABgene Adhesive Plate Seals&amp;nbsp;&amp;nbsp;</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>AB0580</td><td></td></tr><tr><td>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><td>Verso cDNA synthesis Kit</td><td>Thermo-Fisher Scientific (Waltham, MA, USA)</td><td>AB1453B</td><td></td></tr><tr><td>ViiA 7 Real-Time PCR System</td><td>Applied Biosystems (Foster City, CA, USA)</td><td>4453536</td><td></td></tr><tr><td>Water-Ultra Pure</td><td>Quality Biological, INC. (Gaithersburg, MD, USA)</td><td>351-029-101</td><td></td></tr><tr><td>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></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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

13.3K 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

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

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

遗传学

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

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 ...

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

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

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

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

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

基于探针实时荧光定量PCR途径的小分子RNA定量检测

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

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

用于呼吸道病毒检测的多重 RT-qPCR 检测

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public&#39;s health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

作者聚焦:呼吸道病毒多重检测的进展 

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

开发用于检测SARS-CoV-2、甲型/乙型流感和MERS-CoV的多重实时RT-qPCR检测

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...

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

摘要

探索更多视频

此视频中的章节

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

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

流感病毒的高通量检测方法

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

基于探针实时荧光定量PCR途径的小分子RNA定量检测

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

开发用于检测SARS-CoV-2、甲型/乙型流感和MERS-CoV的多重实时RT-qPCR检测

开发用于检测SARS-CoV-2、甲型/乙型流感和MERS-CoV的多重实时RT-qPCR检测

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

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

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

流感病毒的高通量检测方法

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

基于探针实时荧光定量PCR途径的小分子RNA定量检测

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

开发用于检测SARS-CoV-2、甲型/乙型流感和MERS-CoV的多重实时RT-qPCR检测

开发用于检测SARS-CoV-2、甲型/乙型流感和MERS-CoV的多重实时RT-qPCR检测

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