We give special thanks to Ezra Lyon, Eliot Zhu, Michele Wing, Esko Kautto and Eric Samorodnitsky for technical support. We would also like to thank Jenny Badillo for her administrative support for our team. We acknowledge the Ohio Supercomputer Center (OSC) for providing disk space, processing capacity, and support to run our analyses. We thank the Comprehensive Cancer Center (CCC) at The Ohio State University Wexner Medical Center for their administrative support of this work. S.R. and Team are supported by the American Cancer Society (MRSG-12-194-01-TBG), a Prostate Cancer Foundation Young Investigator Award, NHGRI (UM1HG006508-01A1), Fore Cancer Research Foundation, American Lung Association, and Pelotonia.

注意:此协议说明了同时处理和4个样品的分析。这种方法是用RNA从细胞中分离，新鲜冷冻组织和福尔马林固定，石蜡包埋的组织的（FFPE）兼容。该协议始于50 - 从RNA输入每个样本的1000纳克（250纳克推荐）。 1. rRNA的消耗和RNA议事碎片<ol><li> rRNA的消耗 <ol><li>在室温下除去洗脱，素，片段组合，rRNA的去除组合，rRNA的结合缓冲液和重悬浮缓冲液从-20℃并解冻。从4℃除去的洗脱缓冲液，rRNA的去除珠和RNA / cDNA的特定顺磁性珠粒，并把到室温。 </li><li>添加0.25微克RNA​​对PCR管。稀释用不含核酸酶的超纯水的总RNA，以10微升总终体积。添加5微升的rRNA结合缓冲液，以每管再加入的rRNA去除混合5微升，轻轻pipettË向上和向下混合。在与盖的热循环仪的地方管预加热至100℃，程序热循环仪于68℃5分钟以变性RNA。 </li><li> RNA变性后，从热循环仪取出试管并在室温下孵育1分钟。涡rRNA基因去除珠管大力重悬珠。添加35微升的rRNA去除珠新管和转移RNA变性反应（20微升）添加到含有的rRNA去除珠管。 </li><li>调整吸管45微升和吸管迅速向上和向下20X混合。在RT孵育管1分钟。然后放置在磁性支架管在室温下放置1分钟。转移上清液至新标记的PCR试管中在磁性支架再次将这些试管在室温下放置1分钟。这将确保没有珠粒转移。转移上清至新标记的PCR管。 </li><li>涡流的RNA / cDNA的特定顺磁珠，并添加99微升的珠每管。轻轻吸管整个音量上下10倍混合。如果有降级-RNA起，加193微升充分混合的RNA / cDNA的顺磁性的珠子，每管。孵育在室温下进行15分钟。然后将试管在室温下的磁性支架5分钟。取下并丢弃上清液。 </li><li>加入200μl新鲜配制的70％乙醇（乙醇）的。保持磁架管和照顾到不打扰珠。在室温下孵育30秒，然后取出并丢弃上清液。允许管放置在室温下5 - 10分钟进行干燥。 </li><li>离心机解冻室温洗脱缓冲液在600×g下5秒。添加11微升洗脱缓冲液对每个管和轻轻吸取上下10倍混合。在RT孵育管2分钟。放置在磁性支架管在室温下5分钟。将上清转移的8.5μl到新标记的PCR试管中。 </li></ol></li><li> rRNA基因RNA耗尽的碎片 <ol><li>加入8.5微升埃尔UTE，8.5微升黄金和8.5微升片段组合，以每管。吸管轻轻向上和向下10倍混合。地方管在与下面的程序热循环仪:预加热盖至100℃，94℃8分钟，然后4℃保持。 注意:此步骤是为了产生155碱基对的平均插入片段大小。如果RNA样品的平均片段大小低于200基点，跳过此步骤并继续执行第一链cDNA合成。 </li></ol></li></ol> 2. cDNA合成<ol><li> 合成第一链cDNA <ol><li>从-20°C取出第一链合成的混合，在室温下解冻。 </li><li>预编程热循环仪，使用以下设置:预热盖选项和设定为100℃，然后25℃10分钟，42℃15分钟，70℃15分钟，并保持在4℃下。程序保存为"合成第一链。" </li><li>在600＃离心机解冻-第一链合成混合管215;克5秒。将1微升逆转录酶与9微升第一链合成混合。 </li><li>添加8微升第一链合成的和逆转录酶混合到每个管中，轻轻地吸取上下6倍混合。使用固定速度的迷你台式离心机在600 XG 4秒的离心管中，使液体底部。将管在热循环仪，并选择合成第一链。当热循环仪达到4℃，除去管和立即进行合成第二链cDNA。 </li></ol></li><li> 合成cDNA第二链 <ol><li>预热热循环仪，以16℃下与盖子预先加热至30℃。解冻第二链预混并在冰上再悬浮缓冲。在事先从4℃除去的顺磁性珠的瓶子，静置至少30分钟，以使它们到室温。 </li><li>添加5μl的重悬浮缓冲液，以每个PCR管中。离心机第二链预混600×g下5秒，并添加20微升到每个PCR管中。地方管在16℃，1小时预热的热循环仪。当完成温育后，从热循环仪取出，并允许管来室温。 </li><li>涡流的顺磁性珠粒，直到它们被充分分散。添加90微升充分混合的顺磁珠到每个管中。轻轻吸取整个音量上下10倍混合。在室温下孵育管15分钟。在室温下的磁性支架5分钟地方管。 </li><li>取下并丢弃135微升上清液然后离开磁力架管进行乙醇洗。加入200μl新鲜制备的80％的乙醇，以每管，而不会干扰所述珠，然后在室温下孵育30秒。取下并丢弃所有的上清液从每个。重复一共有两个80％的乙醇洗涤。 </li><li>让试管在室温下静置5 - 10分钟以在磁性支架干燥。离心解冻室temperatur在600×克5秒ê悬浮缓冲液。从磁性底座取出PCR管。添加17.5微升悬浮缓冲液到每个PCR管，轻轻吸取整个音量上下10倍到调匀。孵育管在室温下2分钟。 </li><li>在室温下5分钟的磁性支架地方管，然后转移15微升上清液（双股cDNA）至0.2ml的PCR条管中。 注意:这是如cDNA可被储存在-20℃下长达7天的安全停止点。 </li></ol></li></ol> 3.文库制备<ol><li> 腺苷酸3&#39;端 <ol><li>在室温下拆下-20°C和解冻A尾组合。预编程热循环仪使用以下设置:选择预热盖选项和设定为100℃，然后将在37℃30分钟，70℃5分钟，并保持在4℃。这个程序保存为"ATAIL70"。 </li><li>加入2.5微升再悬浮的缓冲到每个管中。然后加入12.5微升解冻A尾组合。轻轻吸取整个音量上下10倍到调匀。将管在热循环仪，并选择ATAIL70。当热循环仪的温度是在4℃下，从热循环仪取出PCR管并立即进行结扎适配器。 </li></ol></li><li> 结扎适配器 <ol><li>除去合适的RNA适配器管，从-20℃停止连接缓冲液和重悬浮缓冲液并在室温下解冻。直到指示在协议中这样做，不要从-20°C除去连接混合物管。从4℃除去的顺磁性珠的瓶子，静置至少30分钟，使至室温。 </li><li>预加热热循环仪，以30℃，并选择预加热盖选项和设定为100℃。离心600×克5秒解冻RNA适配器管。在使用前立即从-20℃取出连接混合物管存储。 </li><li>添加2.5微升的再悬浮缓冲液中向每个样品管中。加入2.5微升连接混合物。返回连接混合物管-20°C使用后立即存储。添加2.5微升解冻的RNA适配器索引的向每个样品管中。轻轻吸取整个音量上下10倍到调匀。 </li><li>使用固定速度的迷你台式离心机在600×g的时间为4秒离心管中。管放置在预热的热循环仪。盖上盖子，在30℃下孵育10分钟。 </li><li>除去从热循环仪管和添加5微升停止连接缓冲液到每个管灭活结扎。轻轻吸取整个音量上下10倍到调匀。 </li><li> 2.2.4，使用42微升的混合顺磁珠，并丢弃79.5微升上清 - 如2.2.3所述重复洗。与在磁性支架上的管，让在室温下进行5样品风干 - 10分钟。 </li><li>拆下的PCR条管磁架，并添加52.5微升悬浮缓冲液每管。轻轻吸取整个音量上下10倍到调匀。在室温下孵育管2分钟。放置在RT磁性支架上的管5分钟，或直至所述液体是明确的。转移上清液加入50μl来自每个管新0.2毫升的PCR条管中。注意不要打扰珠。 </li><li> 2.2.4，用50微升的混合顺磁珠，并丢弃95微升上清 - 如2.2.3所述重复洗。与在磁性支架上的管，让在RT样品风干5 - 10分钟。 </li><li>从磁性底座上卸下的PCR条管和添加22.5微升重悬浮缓冲液到每个管中。轻轻吸取整个音量上下10倍到调匀。 </li><li>孵育在RT管2分钟。放置在磁性支架上的管，在室温下放置5分钟或直至液体是明确的。转移20微升上清液从每个管到一个新0.2毫升PCR条管。注意不要打扰珠。这是一种安全的停止点和cDNA可以储存在-20℃达7天。 </li></ol></li></ol> 4.图书馆放大<ol><li> 丰富DNA片段 <ol><li>在室温下取出PCR主混合物，PCR引物鸡尾酒和再悬浮缓冲从-20°C的存储和解冻。除去从4℃贮存的顺磁性珠的瓶中，静置至少30分钟，使至室温。 </li><li>预编程热循环仪使用以下设置:选择预热盖选项和设定为100℃，然后在98℃设定的初始变性在98℃，30秒，变性的15个循环持续10秒，退火在60℃下30秒，和延伸72℃30秒，在72℃下的最后一个扩展周期为5分钟，并保持在4℃。程序保存为"PCR"。 </li><li>离心解冻PCR主在600×g下5秒混合和PCR引物的管。添加5微升的解冻PCR引物，以每个样品管中。加入25微升解冻PCR扩增预混每个样品管中。轻轻吸取整个音量上下10倍到调匀。 </li><li>请将加盖PCR条管的预编程热循环仪。盖上盖子，运行PCR程序。当PCR完成后，从热循环仪取出试管，并保持在冰上。 </li><li> 2.2.4，用50微升的混合顺磁珠，并丢弃95微升上清 - 如2.2.3所述重复洗。与在磁性支架上的管，让在室温下进行5样品风干 - 10分钟。 </li><li>从磁性支架移除管和添加32.5微升重悬浮缓冲液到每个样品管中。轻轻吸取整个音量上下10倍到调匀。孵育在室温下进行2分钟。放置在磁性支架上的PCR试管在室温下5分钟，或直至所述液体是明确的。然后换乘30微升苏pernatant新鲜0.2毫升PCR管。 </li><li>使用荧光计7执行cDNA的定量和使用毛细管电泳系统8确定的cDNA质量。 注意:这是一个安全的停止点和cDNA可以储存在-20℃达7天。注:该库被认为是一种RNA测序文库。 后续步骤导致捕获库。 </li></ol></li></ol> 5.杂交，捕获和测序<ol><li> 复用杂交 <ol><li>从-20°C删除自定义水合探头，婴儿床-1 DNA，阻断通用寡核苷酸，以及适配器特异性阻断寡核苷酸和解冻冰。 </li><li>在低绑定1.5mL管中，并结合500 ng的DNA（每个样品125纳克时，复用4个样本），5微升婴儿床-1 DNA（1微克/微升），1UL通用阻断寡核苷酸，0.5微升P7（6个核苷酸）适配器特定阻断寡核苷酸（该量可能需要根据多路复用conditi进行调整项）和0.5微升P7（8核苷酸）适配器特异性阻断寡聚（该量可能需要根据多路复用条件）进行调整。 </li><li>将样品管在真空浓缩器具有面向旋转的相反方向帽打开。在45℃下20分钟，或直至所述液体完全蒸发干燥的内容。 </li><li>干重悬内容与8.5微升2X杂交缓冲液，3.4微升杂交A组份和1.1微升无核酸酶的水。允许再悬浮和涡每2.5分钟10分钟。再悬浮的物质转移至0.2mL的PCR管并在95℃下孵育10分钟在热循环。 </li><li>从热循环仪中删除的杂交样品管，加入2微升重新悬浮在1.5皮摩尔/微升的浓度定制探针。备选地，加入4微升重新悬浮在0.75皮摩尔/微升的浓度定制探针。孵育杂交反应过夜 - 在65℃下（16 24小时）。 注:探头从不同的供应商处购买之间可以使用和制造商的说明应遵循。杂交步骤的持续时间也可以变化。 </li></ol></li><li> 珠制备及捕获 <ol><li>从4℃除去的链霉抗偶联的顺磁珠瓶并在室温下平衡30分钟。稀释10倍的洗涤缓冲液（I，II，III和严格）和2.5倍珠洗涤液以创建1X工作方案。 </li><li>等分试样140微升1×洗涤缓冲液的我成到一个新的1.5毫升管。的1倍严格缓冲热量全部量，并在65℃下1×洗涤缓冲液I的等分试样在至少2小时的热块。 </li><li>等份加入100μl的每捕获蛋白链菌素偶联的顺磁珠到1.5毫升管。放置在磁铁和弃上清。添加每100微升磁珠200μl的珠洗涤缓冲液并涡旋10秒。在磁铁将用于2 - 5分钟或直到上清液是显而易见的。一旦上清液是明确的，放弃它，并重新泥炭一次，总共两次洗涤的。 </li><li>拆除珠子洗涤缓冲液后，加入等体积的珠洗涤液作为初始起点体积（ 即 100微升一个捕获）。重悬并转移到0.2mL的PCR管中。将管磁架为2 - 5分钟或直到上清液是显而易见的。弃上清。 </li><li>既在在65℃的热循环仪的杂交样品和珠，杂交混合物转移到珠管和移液管上下10倍混合。孵育在65℃下45分钟，涡旋，并使用一个固定的速度（6.0 XG）台式微型离心机降速样品4秒。 </li></ol></li><li> 珠洗 <ol><li>从热循环仪中取出捕获管，加入100微升预先加热1×洗涤缓冲液I至管，涡旋10秒以混合。将混合物转移到一个新的低绑定1.5mL管中。放置管中磁分离齿条和允许2 - 5分钟进行分离，或直至上清液是明确的。 ðiscard上清。 </li><li>加入200μl预热1X严格的洗涤液和移液器上下10次混合。孵育在65℃下5分钟。把试管磁分离架上，并允许2 - 3分钟分离或直到上清液是显而易见的。弃上清。重复严格洗涤一次，总共两次洗涤的。 </li><li>加入200μl室温1×洗涤缓冲液I和涡旋2分钟以混合。放置管在磁性分离架上并允许2 - 5分钟进行分离，或直至上清液是明确的。弃上清。 </li><li>加入200μl室温1×洗涤缓冲液II和涡旋1分钟以混合。放置管在磁性分离架上并允许2 - 5分钟进行分离，或直至上清液是明确的。弃上清。 </li><li>加入200μl室温1×洗涤缓冲液III和涡旋30秒以混合。放置管中磁分离机架允许2 - 5分钟进行分离，或直至上清液清楚了。弃上清。 </li><li>从磁分离机架上卸下该管，并添加20微升不含核酸酶的水重悬珠子。吹打向上和向下10倍调匀。 </li></ol></li><li> 拍摄后的PCR扩增 <ol><li>从4℃除去顺磁珠，在室温下平衡30分钟。 </li><li>在RT除去-20℃并解冻的热启动PCR预拌（2×）和PCR引物混合，然后置于冰上。 27.5微升2倍热启动PCR预拌与PCR引物1的2.75微升至2.75微升PCR引物2相结合的图书馆准备扩增预混（这些卷1杂交库加过量10％）。 </li><li>设置通过加入珠20微升加与文库扩增主混合物的30微升的50微升的总体积捕获的DNA在PCR管进行反应。帽筒正确，涡旋混合。 4秒的离心管使用一个固定的速度迷你卡在6.0×g的LE-离心机。 <ol><li>设置下述PCR程序: - 在98℃变性12个周期为15秒，退火65选择预热盖选项和设定为100℃，然后将初始变性在98℃45秒，10 ℃30秒和延伸72℃60秒，在72℃下的最后一个扩展周期为60秒并保持在4℃。 </li></ol></li><li>从热循环仪取出样本，加入75微升顺珠。拌匀，并在室温孵育15分钟。 </li><li>放置在磁体管在室温下2 - 3分钟，然后除去上清液。通过加入200微升80％乙醇，温育30秒，然后除去上清液洗上磁珠。重复总共两次80％洗涤。 </li><li>在RT孵育5 - 10分钟，以允许珠粒干燥。不要过度干燥，开裂。珠悬浮在22微升的Tris-EDTA pH值8.0（1X TE解决方案），并允许为洗脱3分钟。将样品在磁铁3 - 5分钟，日恩转移20微升洗脱的产品来新低绑定1.5mL管中，以确保没有珠结转。 </li><li>执行使用荧光计7捕获的cDNA的定量和使用毛细管电泳系统8确定捕获的cDNA质量。 </li></ol></li><li> 桌面序时的加载过程9 <ol><li>稀捕获cDNA文库利用10毫摩尔Tris-氯pH 8.5的具有0.1％Tween 20的解冻10N的NaOH和杂交缓冲在冰上4 1nM的最终浓度。使用前约30分钟，在解冻水RT桌面序V2试剂盒盒1。不要填写上面的最大加载线10。请注意，这是一个测序平台的具体程序和政策有可能根据制造商的说明而变化。 </li><li>通过在离心管（始终准备好新鲜）20微升10 N氢氧化钠980微升核酸游离水结合准备的0.2N氢氧化钠1毫升。通过稀释PhiX库（库控制）至4纳米组合2微升10与3微升的10mM三Cl pH 8.5的纳米库控制的具有0.1％Tween 20。 </li><li> 5微升4nm的图书馆与5微升0.2N的氢氧化钠和旋涡简要相结合的混合最终变性图书馆和图书馆的控制。使用固定速度小型台式离心机在6.0×g下4秒离心管中。孵育在RT 5分钟以变性库。 </li><li>添加990微升预冷的杂交缓冲液含有10微升变性库的管中。这导致了20 PM库。标记变性20 PM库与日期，并可以在-20℃储存长达3周。 </li><li>混合375微升20日下午库控制的225微升预冷杂交缓冲液稀释库控制导致12.5分库控制。倒置几次到该溶液中混合。 </li><li>结合594微升变性最终库6微升下午12时05分变性库控制和旋涡混合的。将结合SAMP乐库和磁带库控制预留冰上直到样品准备加载到桌面序试剂盒。 </li></ol></li></ol> 6.数据分析<ol><li> 序列质量评估 <ol><li>计算使用序列质量评估11原始序列数据（FASTQ文件）的质量。 注意:此步骤有助于评估数据之前，进一步进行下游分析。该软件具有内置参数运行，并产生一组度量为每个FASTQ文件。 </li></ol></li><li> 对准 <ol><li>对齐序列读取（FASTQ文件）的参考人类基因组hg19并使用Tophat2 12转录（2.0.10版本），同时提供成绩单称为一个GTF文件。输出是在所谓的BAM文件的二进制对准格式的形式。 </li><li>执行包括排序，并使用Samtools 13（版本0.1.19索引后处理步骤）的BAM文件。执行重复的标记，重新排序SAM，插入尺寸计算，并使用工具皮卡德14（1.84版）增加或更换读取组。 </li></ol></li><li> RNA测序质量评估 <ol><li>计算使用RNA测序质量评估RNA测序数据的一系列的质量控制指标。输入到这个软件是从Tophat2对准一个BAM文件15。输出是一个HTML文件，该目录总读取次数，重复的，映射的读百分比和rRNA的百分比， 等等 ，等等。 </li></ol></li><li> 变异召唤 <ol><li>使用STAR（版本2.4.0）16对齐，然后调用单核苷酸变异使用GATK的（版本3.3-0）HaplotypeCaller 17。遵循GATK BAM后处理步骤和过滤标准标志，并从输出中删除误报。 </li></ol></li><li> 基因表达 <ol><li>计算基因表达USING袖扣从燕尾服套装软件18（版本2.1.1）。 注意:输入来自Tophat2对齐工具BAM文件。输出是在同种型，基因和转录物水平，其中，表达作为FPKM（每百万映射读取片段每千碱基）计算产生的。 </li></ol></li><li> 融合呼叫 <ol><li>呼叫使用ChimeraScan 19（0.4.5版本），高顶礼帽融合20个自定义和TRUP 21每个样品的融合。注释使用Oncofuse 22（版本1.0.9b2）域的融合。 </li></ol></li></ol>

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B., 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=Quantitative+gene+profiling+of+long+noncoding+RNAs+with+targeted+RNA+sequencing.">Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing.</a> Nat Methods. 12, 339-342 (2015).</li><li>Cieslik, 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=The+use+of+exome+capture+RNA-seq+for+highly+degraded+RNA+with+application+to+clinical+cancer+sequencing.">The use of exome capture RNA-seq for highly degraded RNA with application to clinical cancer sequencing.</a> Genome Res. , (2015).</li><li>Cabanski, C. R., 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=cDNA+hybrid+capture+improves+transcriptome+analysis+on+low-input+and+archived+samples.">cDNA hybrid capture improves transcriptome analysis on low-input and archived samples.</a> J Mol Diagn. 16, 440-451 (2014).</li><li>Costa, C., Gimenez-Capitan, A., Karachaliou, N., Rosell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+screening:+from+the+RT-PCR+to+the+RNA-seq.">Comprehensive molecular screening: from the RT-PCR to the RNA-seq.</a> Transl Lung Cancer Res. 2, 87-91 (2013).</li><li>Zhao, W., 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=Comparison+of+RNA-Seq+by+poly+(A)+capture,+ribosomal+RNA+depletion,+and+DNA+microarray+for+expression+profiling.">Comparison of RNA-Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling.</a> BMC Genomics. 15, 419 (2014).</li></ol>

S.R. receives funding from Novartis and Ariad Pharmaceuticals for conduct of clinical trials. S.R. immediate family members own stock in Johnson and Johnson.

RNA测序Capture是RNA测序和芯片之间的中间策略评估转录的选定部分接近。捕获的优点包括在桌面序器，高吞吐量，和基因组改变的检测降低的成本，快速的周转时间。可适于表征非编码RNA 23，检测单核苷酸的方法变体4-6，检查RNA剪接，并确定基因融合或结构重排24。此外，这种方法可应用于已经经过用福尔马林固定并包埋在石蜡块24,25临床或处理的样本。 <p class=...

Whole transcriptome or RNA sequencing (RNAseq) is an unbiased sequencing method to assess all RNA products. The goal of targeted RNAseq (Capture) is a focused evaluation of selected transcripts with increased sensitivity, dynamic range, reduced cost or scale, and increased throughput compared to standard RNAseq. Similar to standard RNAseq, targeted enrichment approaches can be used to evaluate gene expression, multiple RNA species such as mRNA, microRNA (miRNA), lncRNA1, other noncoding RNAs2, gene fusions3, and mutations4-6.
Capture involves hybridization of complementary oligonucleotides to enrich cDNA libraries for sequencing. The rationale for RNAseq Capture is similar to microarray approaches where complementary oligonucleotides or probes are hybridized to samples and then measured for relative abundance. For microarray technologies, expression is based on relative signal measured for transcripts binding to these probes. Microarrays are thus limited by range, potential background noise from non-specific binding, and cross-hybridization of probes. Furthermore, arrays have limited dynamic range for low and highly expressed transcripts compared to RNAseq1. Microarrays are widely utilized due to their reduced cost and high throughput capacity compared to RNAseq. 
Here, we demonstrate a method for RNAseq Capture that offers a middle ground between RNAseq and microarray approaches for evaluating the transcriptome. RNAseq Capture has intermediate throughput, greater dynamic range and sensitivity, and is scaled for fast turnaround on desktop sequencers. RNAseq Capture also requires reduced computational resources in terms of storage space and data processing.

<table><tbody><tr><td>Thermomixer R</td><td>Eppendorf</td><td>21516-166</td><td></td></tr><tr><td>Centrifuge 5417R</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>miRNeasy Mini Kit</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>Molecular Biology Grade Ethanol</td><td>Sigma Aldrich</td><td>E7023-6X500ML</td><td></td></tr><tr><td>Thermoblock 24 x 1.5 ml</td><td>Eppendorf</td><td>21516-166</td><td></td></tr><tr><td>MiSeq Reagent Kit v2 (300-cycles)</td><td>Illumina</td><td>MS-102-2002</td><td></td></tr><tr><td>MiSeq Desktop Sequencer</td><td>Illumina</td><td></td><td></td></tr><tr><td>PhiX Control v3</td><td>Illumina</td><td>FC-110-3001</td><td></td></tr><tr><td>TruSeq Stranded Total RNA Kit with RiboZero Gold SetA</td><td>Illumina</td><td>RS-122-2301</td><td></td></tr><tr><td>25 rxn xGen&amp;reg; Universal Blocking Oligo - TS-p5</td><td>IDT</td><td>127040822</td><td></td></tr><tr><td>25 rxn xGen&amp;reg; Universal Blocking Oligo - TS-p7(6nt)</td><td>IDT</td><td>127040823</td><td></td></tr><tr><td>25 rxn xGen&amp;reg; Universal Blocking Oligo - TS-p7(8nt)</td><td>IDT</td><td>127040824</td><td></td></tr><tr><td>Agencourt&amp;reg; AMPure&amp;reg; XP - PCR Purification beads&amp;nbsp;</td><td>Beckman-Coulter</td><td>A63880</td><td></td></tr><tr><td>Dynabeads&amp;reg; M-270 Streptavidin</td><td>Life Technologies</td><td>65305</td><td></td></tr><tr><td>COT Human DNA, Fluorometric Grade, 1 mg</td><td>Roche Applied Science</td><td>05480647001</td><td></td></tr><tr><td>Qubit&amp;reg; Assay Tubes&amp;nbsp;</td><td>Life Technologies</td><td>Q32856</td><td></td></tr><tr><td>Qubit&amp;reg; dsDNA HS Assay Kit</td><td>Life Technologies</td><td>Q32851</td><td></td></tr><tr><td>SeqCap&amp;reg; EZ Hybridization and Wash Kits&amp;nbsp; (24 or 96 reactions)</td><td>Roche NimbleGen&amp;nbsp;</td><td>05634261001 or 05634253001&amp;nbsp;</td><td></td></tr><tr><td>Qubit&amp;reg; 2.0 Fluorometer&amp;nbsp;</td><td>Life Technologies</td><td>Q32866</td><td></td></tr><tr><td>10 x 2 ml IDTE pH 8.0 (1x TE Solution)</td><td>IDT</td><td></td><td></td></tr><tr><td>Tween20 BioXtra</td><td>Sigma</td><td>P7949-500ML</td><td></td></tr><tr><td>Nuclease Free Water</td><td>Life Technologies</td><td>AM9937</td><td></td></tr><tr><td>C1000 Touch&amp;trade; Thermal Cycler with 96&amp;ndash;Well Fast Rection Module</td><td>Biorad</td><td>185-1196</td><td></td></tr><tr><td>SeqCap EZ Hybridization and Wash Kits</td><td>Roche Applied Science</td><td>05634253001</td><td></td></tr><tr><td>SuperScript II Reverse Transcription 200 U/&amp;#956;l</td><td>Life Technologies</td><td>18064-014</td><td></td></tr><tr><td>D1000 ScreenTape</td><td>Agilent Technol. Inc.</td><td>5067-5582</td><td></td></tr><tr><td>Agencourt RNAClean XP - 40 ml</td><td>Beckman Coulter Inc</td><td>A63987</td><td></td></tr><tr><td>RNA ScreenTape</td><td>Agilent Technol. Inc.</td><td>5067-5576</td><td></td></tr><tr><td>RNA ScreenTape Ladder</td><td>Agilent Technol. Inc.</td><td>5067-5578</td><td></td></tr><tr><td>RNA ScreenTape Sample Buffer</td><td>Agilent Technol. Inc.</td><td>5067-5577</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>72068-100ML</td><td></td></tr><tr><td>DynaBeads MyOne Streptavidin T1</td><td>Life Technologies</td><td>65602</td><td></td></tr><tr><td>DYNAMAG -96 SIDE EACH</td><td>Life Technologies</td><td>12331D</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C2432-1L</td><td></td></tr><tr><td>KAPA HotStart ReadyMix</td><td>KAPA Biosystems</td><td>KK2602</td><td></td></tr><tr><td>NanoDrop 2000 Spectrophotometer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>My Block Mini Dry Bath</td><td>Benchmark</td><td>BSH200</td><td></td></tr><tr><td>D1000 Reagents</td><td>Agilent Technol. Inc.</td><td>5067- 5583</td><td></td></tr><tr><td>Vacufuge Plus</td><td>Eppendorf</td><td>022829861&amp;nbsp;</td><td></td></tr></tbody></table>

targeted rna sequencing assay to characterize gene expression and genomic alterations

RNA测序（RNA测序）是可以被用于检测和表征的基因表达，突变，基因融合，和非编码RNA一个通用的方法。标准RNA测序需要30 - 亿测序读出并可以包括多个RNA产物如mRNA和非编码RNA。我们证明有针对性的RNA测序（捕获）如何允许在使用桌面序选定的RNA产品聚焦研究。 RNA测序采集可以表征可能以其它方式使用传统的RNA测序方法错过没有注释，低或瞬时表达成绩单。在这里，我们描述了从细胞系，核糖体RNA耗尽，cDNA合成，制备条形码库，杂交和靶向转录物的捕获和多重测序上的桌面序的RNA的提取。我们也勾勒出计算分析管道，其中包括质量控制评估，校准，检测融合，基因表达定量和单国统会的鉴定leotide变种。该测定允许有针对性的转录测序来表征的基因表达，基因融合，和突变。

在RNA测序捕捉的概略突出关键步骤示于图1。与已知的突变四癌细胞系被用来证明RNA测序捕捉技术的有效性（K562与ABL1融合，LC2与RET融合，EOL1与PDGFRalpha融合和RT- 4 FGFR3融合）。这四个样品汇集在一起​​，并进行测序2倍于台式机音序器，生成FASTQ文件100个基点的读取。 1）质量控制评估，2）对准至人类转录，3）基因表达的定量，4）?...

Watch this Scientific Journal Video about Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations at JoVE.com

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

targeted-rna-sequencing-assay-to-characterize-gene-expression-genomic

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.3K 次观看.  The Ohio State University. We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

视频: Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

利用RNA测序来检测新剪接变异体在相关耐药<em&gt;体外</em&gt;癌症模型

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

科研

癌症研究

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

遗传学

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

组合 miRNA 治疗调节细胞周期和血管生成的分析

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的 离体 消融来定义肿瘤发生的基因功能

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

HDAC 抑制剂介导的 B 细胞 rna 和基因的基因组分析--诱导进行类开关 DNA 重组和血浆细胞分化

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

免疫与感染

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

医学

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

生物学

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

使用 scRNA-Seq 和 scATAC-Seq 对视网膜基因表达和染色质可及性进行多重分析

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

神经科学

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

在EML造血前体细胞调节自我更新和分化的关键因素，通过RNA测序分析鉴定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

有针对性的RNA测序分析表征基因表达和基因组改变

摘要

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此视频中的章节

利用RNA测序来检测新剪接变异体在相关耐药

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

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HDAC 抑制剂介导的 B 细胞 rna 和基因的基因组分析--诱导进行类开关 DNA 重组和血浆细胞分化

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

使用 scRNA-Seq 和 scATAC-Seq 对视网膜基因表达和染色质可及性进行多重分析

在EML造血前体细胞调节自我更新和分化的关键因素，通过RNA测序分析鉴定

用 RNA原位杂交法对形态学组织环境中的遗传变异、短靶点和突变进行可视化

有针对性的RNA测序分析表征基因表达和基因组改变

摘要

探索更多视频

此视频中的章节

利用RNA测序来检测新剪接变异体在相关耐药

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

组合 miRNA 治疗调节细胞周期和血管生成的分析

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的 离体 消融来定义肿瘤发生的基因功能

HDAC 抑制剂介导的 B 细胞 rna 和基因的基因组分析--诱导进行类开关 DNA 重组和血浆细胞分化

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

使用 scRNA-Seq 和 scATAC-Seq 对视网膜基因表达和染色质可及性进行多重分析

在EML造血前体细胞调节自我更新和分化的关键因素，通过RNA测序分析鉴定

用 RNA原位杂交法对形态学组织环境中的遗传变异、短靶点和突变进行可视化

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的离体消融来定义肿瘤发生的基因功能