Name: using rna-sequencing to detect novel splice variants related to drug resistance in in vitro cancer models
Uploaded: 2016-12-09T15:00:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models at JoVE.com

The authors would like to acknowledge prof. J.J. McGuire, prof. R. Kofler and dr. K. Quint for providing the resistant cell lines used in this work. The study has been founded by grants from Cancer Center Amsterdam (CCA) Foundation (to JC, EG and RS), the KiKa (Children Cancer-free grant for AW) foundation, the Law Offices of Peter G. Angelos Grant from the Mesothelioma Applied Research Foundation (to VEG and EG), Associazione Italiana per la Ricerca sul Cancro (AIRC), Istituto Toscano Tumori (ITT), and Regione Toscana Bando FAS Salute (to EG).

1.通过细胞毒性分析耐药性型材的表征<ol><li> 白血病细胞株培养 <ol><li>保持亲T细胞在10ml RPMI-1640培养基含有ALL细胞系CCRF-CEM（CEM-WT），以及其药物抗性亚系，包括CEM / R30dm，CEM-R5和CEM-C3，25 平方厘米2.3μM叶酸补充有10％胎牛血清和100单位/ ml青霉素G和100μg/ ml链霉素。 </li><li>培养在5％CO 2培养箱将细胞在湿润气氛中，在37℃。 </li><li>允许2之间细胞生长的浓度- 3×10 6细胞/毫升。 </li><li>每周分裂细胞两次以0.3×10 6细胞/ ml的初始浓度（ 例如，如果细胞浓度为3×10 6个细胞/ ml，在由9毫升新鲜培养基的新烧瓶传送1毫升细胞悬浮液）。经过连续20代丢弃文化。 </li></ol></LI&gt; <li> 胰腺癌细胞株培养 注:保持在75cm 2的培养瓶中的人胰腺癌细胞株的Panc-1在具有高葡萄糖和L-谷氨酰胺补充有10％胎牛血清和100单位/ ml青霉素G和100μg/ ml链霉素10毫升DMEM培养基。耐药变种，PANC-1R，是在含有1μM的吉西他滨溶解在无菌水中的相同培养基中培养。在24提供进一步的细节。 <ol><li>培养在5％CO 2培养箱将细胞在37℃。 </li><li>分裂细胞，每2 - 1天以1:5的比例时，细胞达到约90％汇合。 </li><li>分裂的细胞，用磷酸盐缓冲盐水（PBS）洗两次，每75cm 2的培养瓶中加入1ml胰蛋白酶/ EDTA中，并在37℃孵育3分钟。 </li><li>再加9毫升培养基和收获分离的细胞在15毫升管。在一个新的烧瓶CONTA种子2毫升细胞悬浮液进不去8毫升网上平台。经过连续20代丢弃文化。 </li></ol></li><li> MTT法对白血病细胞 <ol><li>预先制备的MTT溶液:溶于10毫升的PBS 500毫克MTT甲和搅拌（避光）用磁力搅拌器搅拌约1小时。消毒用0.22微米过滤器的溶液。注:该解决方案可以在-20℃储存在10毫升等分试样。解冻后直接避光。 </li><li>提前准备酸化异丙醇:加50毫升2 M盐酸至2.5升异丙醇。注意:使用前在室温下存放的解决方案，至少一个月。如果异丙醇没有被正确酸化，它可能形成具有介质沉淀并危及光度法读数。 </li><li>准备一个单独的96孔平底"第0天"（对照）板，以确保生长抑制的更准确的估计:奉献每个细胞系3至6个孔中，添加30微升的增长平台和120微升每孔150微升生长培养基的细胞悬浮液（8000个细胞）的对应于空白（无细胞）的孔中。从步骤1.3.9进行到步骤1.3.13本条以测量光密度（OD）。注:在4中提供进一步的细节。 </li><li>准备96孔平底试验板:奉献30口井，以药物的浓度（10个浓度，每个一式三份），10口井，以控制细胞和10个孔，以控制无细胞（空白）中，并准备地塞米松的一种药物稀释范围（地塞米松），使用二甲基亚砜作为溶剂。 注:对于CEM-WT细胞的地塞米松稀释范围为2微米和0.97纳米。对于CEM / R30dm，CEM-R5和CEM-C3细胞的地塞米松稀释范围为640微米和0.33纳米之间。 </li><li>添加从每塞米松稀释30微升到96孔板的适当孔。确保包括在一式三份的每个浓度。 </li><li>添加生长培养基的30μl到井的相应对照细胞和150微升生长培养基的对应于空白孔中。 </li><li>收获指数生长的细胞，重悬在其最佳接种浓度。 注意:为了确定最佳的起始细胞浓度时，建议通过在几个浓度接种细胞，并每日测量它为至少4天，以评估在一个96孔板的每个细胞系的细胞系的生长曲线。选择一个播种浓度，防止细胞的过度生长72小时后，因为这将通过饱和OD值影响的实验。对于CEM-WT，CEM-C5和CEM-R5的最佳接种浓度为8000个/孔，而CEM / R30dm是5,000个细胞/孔。 </li><li>添加120微升细胞悬液，以每孔含有任一药液或生长培养基（孔对应于控制单元）。填用150微升的PBS板的空外孔中以保证良好的湿度在所述板孵育第Ë板在37℃，5％的CO 2在细胞培养孵化72小时。 </li><li>添加15微升MTT溶液的各孔中，摇动板5分钟用板摇床最多900摇动/分钟。 </li><li>放置板背面，在37℃，5％的CO 2在细胞培养培养箱孵育另外4 - 6小时。 </li><li>添加150微升酸化异丙醇至每个孔，并用多道移液器拌匀彻底悬浮所有甲晶体。开始与空白孔，并确保在进行到板的另一行之前很好地漂洗提示。 </li><li>孵育在室温下（RT）的板10分钟。 </li><li>用酶标仪，确定的OD在540nm和720nm处通过校正背景外径，以确保准确的测量。然后在电子表格文件保存数据和分析4。 </li></ol></li><li> SRB法对胰腺癌细胞 <ol><li>以0.4％的最终浓度溶解在1％乙酸的SRB试剂（重量/体积）。 </li><li>在50％的最终浓度溶解在超纯水中三氯乙酸（TCA）（重量/体积）。 </li><li>溶解在超纯水中的Tris（羟甲基）氨基甲烷在10毫最终浓度。 </li><li>准备一个单独的96孔平底"第0天"控制盘，以确保生长抑制的更准确的估计:种子6个孔在适当的接种浓度在100μl培养基中指数期生长的细胞和100μl的培养基只添加到对应于井空白。在37℃用5％CO 2孵育过夜，以确保细胞在板的适当的粘合性。然后100微升中添加到所有井，并继续步骤1.4.8 - 1.4.16本节。 </li><li>准备96孔平底试验板:种子细胞在适当的密度生长在指数生长期中，一式三份在96孔平底板在100μl我的通过使用多通道移液管dium。 注意:为了确定最佳的起始细胞浓度时，建议通过在几个浓度接种细胞，并每日测量它为至少4天，以评估在一个96孔板的每个细胞系的细胞系的生长曲线。选择一个播种浓度，防止细胞的过度生长72小时后，因为这将通过饱和OD值影响的实验。对的Panc-1和PANC-1R的细胞，最优接种浓度为8000细胞/孔。 </li><li>将100μl培养基添加至中只孔中，在37℃用5％CO 2孵育过夜，以确保细胞在板的适当的粘合性。 </li><li>制备为1μM和10nM之间的吉西他滨的药物稀释范围的Panc-1和1mM和100nM为的Panc-1R的细胞。通过使用多通道移液管加入从每个稀释100微升到96孔板的适当孔。确保有一式三份每个浓度。此外，将100μl培养基添加至中只有井和对照细胞。在37℃下孵育，用5％CO 2的72小时。 </li><li>通过使用多通道移液管为至少60分钟，在4℃以沉淀，并在孔的底部固定所述蛋白质添加25μl的冷TCA溶液到孔中并孵育所述板。 </li><li>通过在组织除去培养基并干燥简要地清空板。 </li><li>洗5次用自来水，然后清空板，并让在室温下干燥。 </li><li>通过使用重复-吸管和染色，在室温下15分钟加入每孔50μl的SRB溶液。 </li><li>通过去除污渍SRB空板。 </li><li>洗4次，用1％乙酸再清空板，并让它干燥在室温。 </li><li>添加每孔150μl的Tris溶液通过使用多通道移液管和用于在平板摇床最多3分钟混合到最大的900摇动/分钟。 </li><li>读出的光密度在540nm（或如果492nm处吨他OD值过高）。 </li><li>分析数据。 </li></ol></li><li> 数据分析MTT和SRB测定 <ol><li>计算细胞的OD值在"0天"使用下列公式:OD 第0天 = OD 对照细胞 -平均OD 空白孔 </li><li>根据下列公式计算存活的细胞的百分比为每个药物浓度:％治疗细胞=平均[OD 处理的细胞 -平均OD 空白孔 - OD 第0天 ] / [OD 对照细胞 -平均OD 空白孔 -外径Day0] * 100 </li><li>绘制剂量反应曲线（药物浓度 - 在％生长抑制）。 </li><li>计算使用剂量-响应曲线，该曲线的50％抑制细胞生长的药物（IC 50）的浓度。 </li></ol></li></ol> 2. RNA分离和图书馆准备RNA测序<ol><li> 样品科尔挠度和RNA分离<ol><li>对于CEM细胞:直接从培养基中收获10 6个细胞。 </li><li>对的Panc-1和PANC-1R:除去介质，用PBS洗涤细胞两次，并通过添加胰蛋白酶-EDTA，并在37℃孵育3分钟分离。添加培养基和收获10 6个细胞。 </li><li>降速样品在300 XG 3分钟，除去上清液，用市售的二氧化硅膜离心柱，遵循manufacturer`s协议提取总RNA。 </li><li>通过使用紫外可见分光光度计测定总RNA的浓度和纯度。 注:RNA被认为是高纯度的，如果260纳米/ 280nm吸收比高于1.8。样品可以储存在 - 80℃。 </li><li>评估通过在用溴化乙锭染色的1％琼脂糖凝胶200毫​​微克的样品的电泳总RNA的完整性。 注:两种完整频带的对应于哺乳动物28S和18S个rRNA在约的检测2 kb和1 KB的大小指示良好的总RNA的完整性。 </li></ol></li><li> 测序文库制备 <ol><li>使用2微克每每个样品总mRNA的。根据制造商的说明按照mRNA的文库制备协议。 </li><li>通过使用生物分析系统确定每个库的质量和浓度。池单个样品上在库10 nmol / L的终浓度，并与生物分析仪测量。 </li><li>使用高通量测序系统的单读100个基点模式。 注:&gt; 80碱基的读取长度是必要的识别转录亚型。 </li></ol></li></ol> 3.不同的剪接检测从测序读<ol><li> 对准读取到参考基因组和质量检查 <ol><li>编译使用UCSC表浏览器（25963个基因）在NCBI refGene表中的基因注释（.gtf文件）。 </li><li>演出测序注释感知缺口对准读取使用STAR的参考基因组（GRCh37）。 </li><li>排序和指数与皮卡德工具所产生的调整文件。 </li><li>通过使用RSeQC和samtools执行映射后的质量控制。 </li></ol></li><li> 样品组之间的差异剪接检测 <ol><li>安装Python和numpy的和SciPy的相应版本。下载并安装samtools。下载并安装领结和高顶礼帽， </li><li>添加了Python，领结，高顶礼帽和samtools目录到$ PATH环境变量。下载预建领结指数（hg19）。下载rMATS版本3.0.9。 注意:在图19和34中提供了关于rMATS进一步细节。 </li><li>奔跑根据图5A通过比较每个塞米松抗性细胞系CEM-WT和PANC-1到的Panc-1R在单独MATS检测选择性剪接事件。 </li><li>检测从previo差别剪接事件usly对齐测序读数（.bam文件），使用这样的命令从图5B为CEM-WT与CEM-C3，CEM-WT与CEM-R 5，CEM-WT与CEM / R30dm和PANC1到Panc-运行MATS 1R比较。 注:MATS将创建一个输出文件夹与每个拼接分析事件的类型结果两项.txt文件（SE - 外显子跳跃，A5SS - 替代5&#39;剪接位点，A3SS - 替代3&#39;剪接位点，MEX - 相互排斥的外显子和RI - 内含子保留）:仅基于结计数和与基于结计数结果的第二文件一个文件含有的结果，并读取关于目标。此外，在同一文件夹中生成一个结果概要的附加.txt文件。 </li><li>对于SE，A5SS，A3SS和RI导入到电子表格基于结计数的.txt文件，读取目标。对于MEX进口仅基于结计数.txt文件。 注:MATS输出文件将被上升的P值和分类包含几个参数:基因身份证，基因符号，染色体和链位置，可变剪接片段的基因组坐标，计数以及夹杂物的长度和跳过两个分析样品形式，夹杂物的长度和跳过用于标准化，p值，假发现率表格（FDR ），基于标准化的计数和微分包含分数（平均（IncLevel1）每个样品包含水平 - 平均（IncLevel2））。 </li><li>与FDR &lt;进一步验证（ 例如 ，DDX5和PKM2）10％选择统计学显著的候选基因。 </li><li>可视化与结合基因组浏览器选择性剪接事件（IGV，https://www.broadinstitute.org/igv/），据报道在图5中。 </li></ol></li></ol> 4.验证通过RT-PCR和定量RT-PCR测定法的结果的<ol><li> 引物设计 注意:为了提供使用生物信息学管道中，mRNA反式获得的结果的一个可靠的验证从选择性剪接事件产生的cripts通过使用RT-PCR扩增和PCR产物的琼脂糖凝胶电泳可视化。花青绿色染料如SYBR绿定量RT-PCR分析被用于量化特定剪接变体相对于管家基因。 图7描绘了施加到形象化DDX5基因的外显子12跳跃事件和量化PKM基因的互斥外显子9和外显子10中的策略。 <ol><li>用于RT-PCR测定法，设计引物对退火到位于上游构外显子（外显子10和外显子13）和下游的可变剪接位点（ 图7A）。 注:扩增子大小应涵盖碱基100和800之间，以确保该预测PCR产物在琼脂糖凝胶上的明确分离。引物的退火温度应在55和65℃，GC含量°之间不应超过60％。 </li><li>花青绿测定（ 图7B）。 <ol&gt; <li>检查两个相互排斥的外显子和设计2种引物对每个具体的序列同源性，只有专门检测两个剪接变体中的一个。 注:对于PKM，反向引物退火到外显子共同11到两个变体，而正向引物变体特异性和退火外显子9（PKM1）或外显子10（PKM2）。 </li><li>为了检测DDX5全长基因，退火跳过外显子（外显子12）内的反向引物。 注:对于DDX5ΔEx12变体的特异性检测，反向引物横跨exon11 / exon13的边界。使用相同的正向引物退火组成的外显子10两个反应。 注:扩增子大小应在80和200碱基对之间。 </li></ol></li></ol></li><li> 第一链cDNA合成 <ol><li>设置的分离的RNA，以cDNA的1微克逆转录用200U /微升莫洛尼鼠白血病病毒的（M-MLV）逆向工程＆在其反应缓冲液本身酶1:5稀释用无菌水。添加DTT1μM的，随机六聚体的0.05微克，脱氧核苷酸的混合物（dNTP）1毫米，和核糖核酸酶抑制剂40U /微升。 </li><li>短暂涡旋并孵育反应混合物在37℃下2小时。 </li><li>孵育反应混合物在70℃下进行5分钟以灭活逆转录酶，在冰上转移混合，并通过使用微量离心。样品可以立即使用或储存于 - 20℃。 </li></ol></li><li> RT-PCR反应和琼脂糖凝胶 <ol><li>通过混合12.5微升2×的设置在每各样品在PCR管中进行PCR反应浓缩的PCR mastermix，1.25微升10μM的正向引物和同量为反向引物达25微升无菌水的终体积。 </li><li>加入1μl的cDNA的组合，然后将试管中的热循环。运行该程序如下:初始变性:95℃2分钟。重申下述步骤35个循环:变性95℃25秒;退火52℃35秒;延伸72℃1分钟。在72℃下5分钟，设置最终延伸。 </li><li>将1g琼脂糖溶解在100毫升1×TBE缓冲液制备1％琼脂糖凝胶。添加2.5微升的溴化乙锭的溶液中。注意:溴化乙锭是致癌物质，通过化学通风柜谨慎操作。 </li><li>加载样品和在100伏运行在1×TBE缓冲液的凝胶用于在电泳系统约30分钟。 </li><li>显示用数码相机UV胶并保存图像。 </li></ol></li><li> 定量RT-PCR和数据分析 <ol><li>设置每各样品的花青绿色PCR反应由混合12.5微升2×绿色的PCR反应体系的，2微升5微米正向引物和相同数量的反向引物达15微升无菌水在一PCR管的终体积。制备混合为每个特定的拼接变被检测到（PKM1，PKM2，DDX5全长，DDX5ΔEx12）和管家基因（GUS）。 </li><li>装载在白色96孔板的mastermix并准备每各样品重复。 </li><li>稀释10倍的cDNA在水中，加入5微升到每个组合，然后将盘在热循环。运行该程序如下:初始变性:95℃5分钟。重申下述步骤45个循环:变性95℃，10秒;退火在58℃（对DDX5和DDX5ΔEx12混合）或20秒60℃（对PKM1和PKM2混合）。在72℃，20秒的设定最终延伸。设置通过施加梯度从65到97℃熔化曲线。 </li><li>为了引物组的特异性评估他们的目标，则检查熔解曲线，并验证单峰每各引物组而形成的。 </li><li>计算扩增曲线的二阶导数的值，并导出周期阈值（CT）。 </li><li> CALCULATE中的mRNA剪接的相对表达水平（REL）的变体相比，GUS看家（参考）基因，通过使用"增量的Ct（ΔCT）"的方法的每个样本。其计算公式为:REL = 2 - ΔCT，其中ΔCT=的Ct 目标剪接变异体 -的Ct 参照基因 </li><li>为了量化剪接变体的相对丰度，通过使用公式计算REL比例:比例REL比= REL 剪接变体1 / REL 剪接变体2。 </li></ol></li></ol>

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J., Jansen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+ex+vivo+methotrexate+resistance+in+acute+lymphoblastic+and+myeloid+leukaemia.">Classification of ex vivo methotrexate resistance in acute lymphoblastic and myeloid leukaemia.</a> Br J Haematol. 110 (4), 791-800 (2000).</li><li>Funel, 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=Laser+microdissection+and+primary+cell+cultures+improve+pharmacogenetic+analysis+in+pancreatic+adenocarcinoma.">Laser microdissection and primary cell cultures improve pharmacogenetic analysis in pancreatic adenocarcinoma.</a> Lab Invest. 88 (7), 773-784 (2008).</li><li>Gillet, J. 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=Redefining+the+relevance+of+established+cancer+cell+lines+to+the+study+of+mechanisms+of+clinical+anti-cancer+drug+resistance.">Redefining the relevance of established cancer cell lines to the study of mechanisms of clinical anti-cancer drug resistance.</a> Proc Natl Acad Sci U S A. 108 (46), 18708-18713 (2011).</li><li>Kole, R., Krainer, A. R., Altman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+therapeutics:+beyond+RNA+interference+and+antisense+oligonucleotides.">RNA therapeutics: beyond RNA interference and antisense oligonucleotides.</a> Nat Rev Drug Discov. 11 (2), 125-140 (2012).</li><li>Shen, 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=rMATS:+robust+and+flexible+detection+of+differential+alternative+splicing+from+replicate+RNA-Seq+data.">rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data.</a> Proc Natl Acad Sci U S A. 111 (51), E5593-E5601 (2014).</li></ol>

The authors have nothing to disclose.

这里，我们介绍，结合成熟的细胞毒性筛选技术和功能强大的基于NGS-转录分析，以鉴别与耐药性差异剪接事件的新方法。分光光度测定法是方便和健壮高通量方法，以评估在体外癌症模型药物敏感性和代表第一选择许多实验室进行的细胞毒性筛查。故障诊断以及用于此方法的可能变化被广泛别处4,5-说明。 高通量基因组分析目前用于探索耐药机制主要依靠单核?...

尽管在癌症治疗中，恶性细胞抗化疗，无论是本征或在长时间暴露于药物取得相当大的进展，是治疗失败的广泛白血病和实体瘤1的主要原因。 为了描绘耐药背后的机制， 在体外细胞系模型是由癌细胞对化疗药物抗性的逐步选择开发的。这个过程模仿临床设置中使用的制度，因此允许在有关耐药机制深入调查。其生存的治疗抗性细胞，然后从亲敏感细胞使用细胞生存力/细胞毒性试验2区分开。 在原代细胞的体外药物抗性型材已被证明是显著相关临床化疗反应3。 高通量cytotoxicity测定构成一种方便的方法，以确定在体外药物的敏感性。在此，细胞的存活率是通过评估实例的3- [4,5-二甲基-2-基] -2,5-二苯基四唑鎓溴化物- MTT法4，这是基于某些基材的代谢转化（即四唑盐）为有色产物，从而反映细胞的线粒体活性。可替代地，细胞蛋白质含量可以使用磺基罗丹明B（SRB）测定5来量化。这里，活细胞的数目正比于在分光光度计的适当波长测得的光密度（OD），不需要的 广泛而耗时的细胞计数的过程。通过一定的化疗药物诱导的生长抑制可以基于其中细胞用测试试剂处理，并用未处理的对照细胞的OD相比的孔的OD来计算。剂量反应曲线是OBTA通过绘制药物浓度对相对于对照细胞的活细胞的百分比INED。最后，药物敏感性可以报告相比，未经处理的细胞（IC 50），其导致细胞生长抑制50％的浓度。 耐药背后的机制包括许多不同的异常，如影响药物活性和细胞代谢的决定因素的基因表达的改变。这些分子的病变，包括突变，像差在转录和转录后水平以及干扰表观遗传调控往往影响或药物代谢或细胞凋亡6所涉及的基因。 替代前体mRNA剪接和它的复杂调控最近受到相当关注，因为这可能会决定癌细胞7耐药一种新型的实体。达人基因95％交替地在正常细胞通过此手段剪接紧紧从同一基因产生许多不同的蛋白质同种型调节的过程。选择性剪接经常失调在癌症和多种肿瘤都受到了越来越多的涉及药物代谢（ 即 ，脱氧胞苷激酶，叶酰聚谷氨酸合成酶，或者多药耐药蛋白）6,8-基因的改变的剪接特征。然而，耐药细胞的拼接型材综合分析是痛苦的缺乏。因此，当务之急是制定选择性剪接分析的高通量方法。这将有助于开发更有效的治疗方法。 在过去十年中，新一代测序（NGS）技术的快速发展，丰富了新的见解执政基因表达的调控以及它们在不同的生物过程9作用的分子机制的生物医学研究。 RNA的测序（RNA-SEQ）是一个功能强大的子应用程序在转录的字段NGS。它允许一个全基因组分析的数千同时基因的表达模式的（在质量和数量），是非常适合于新的编码mRNA的表征以及长的非编码RNA，miRNA的，siRNA的和其他小RNA类（ 如小核RNA和piRNA基因）10,11。 RNA测序已超过以往技术的转录组特征（ 例如，Sanger测序及表达微阵列）的许多优点。它不是基于现有基因组注释，它具有高分辨率的单核苷酸水平并且它有用于表达水平估计更宽的动态范围。简言之，将RNA测序实验的基本实验流程包括多聚腺苷酸转录（mRNA）的选择和碎片，然后转化成cDNA，图书馆建设，最后，大规模并行深度测序12,13。由于sequencin的快速下降在过去的几年中，RNA-SEQ G值正在逐步取代其他技术，并正在作出显著努力提高图书馆准备协议。例如，现在有可能通过标记用脱氧尿苷三磷酸（dUTP的）和之前，PCR扩增，消化与尿嘧啶DNA-glycosilase化酶（UDG）标记的链的第二链cDNA保留mRNA转录物的链信息。该方法增强了表达水平14,15的基因注释和估计的准确度。 分析和RNA测序数据的解读需要生物信息学管道16,17内的复杂和强大的计算软件包和处理。首先，原料读通过消除技术和生物文物和丢弃（切边），它没有达到严格的质量要求序列进行质量控制。接着各样品的读取映射到参考基因组和索引入基因级，外显子级，或转录级，以便确定每个类别的丰度。根据不同的应用，精制数据，然后通过统计模型进行等位基因特异性表达，剪接，基因融合和单核苷酸多态性（SNP）12的识别来计算。最后，在选定的水平（ 即，基因表达或剪接）的差分分析可以用来比较不同条件下获得的样品。 不同的剪接分析介绍了两个样本之间拼接网站使用的差异。越来越多的用于这一目的的软件包可以根据不同的统计模型，表演和用户接口18。其中，MATS（成绩单拼接的多因素分析）涌现作为基于贝叶斯统计框架免费提供的，精确的计算工具，旨在检测迪菲从单个或配对末端RNA测序数据rential剪接事件。从对齐（.bam）文件开始，MATS可以检测所有主要类型的选择性剪接事件（外显子跳跃，替代3&#39;剪接位点，替代5&#39;剪接位点，互斥的外显子和内含子保留-也参见图1）。 首先，软件识别读取支持一定剪接事件，例如外显子跳跃，并把它们分类成两种类型。 "夹杂读取"（用于规范剪接事件）映射调查外显子内，并且跨越该特定外显子和两个上游和下游侧翼的外显子之间的连接处。 "跳读"（用于选择性剪接事件）跨越两个侧翼外显子之间的交界处。随后，MATS返回纳入规范化水平为规范和替代的事件和样品或条件之间进行比较值。最终，它计算P值一第二假发现率（FDR）假设两个条件之间的基因的变体比率的差超过为每个拼接事件19,34给定的用户定义的阈值。 以下在用RNA-SEQ结合差别剪接分析，广泛的实验验证以鉴定真阳性候选基因18必要的。定量反转录聚合酶链式反应（QRT-PCR）是由RNA测序分析20获得候选验证最常用的和最佳的方法。本文的目的是提供一种完善的方法来调查在实体瘤和血液恶性肿瘤药耐药相关剪接轮廓。我们的方法利用了与耐药性有牵连的候选基因的验证建立定量RT-PCR方法相结合抗药性癌症的选择细胞系模型的RNA-SEQ基于转录组分析。 5）21,22和甲氨蝶呤（MTX）抗性亚系CEM / R30dm 23。虽然基于GC和MTX的当前疗法的情况下，约90％建立临床益处，GC-耐药性的出现仍然代表具有不明确的分子机理的未解决的问题。以分离抗性的GC-子克隆，CEM-WT细胞在1μM的地塞米松为2〜3周培养。 MTX抗性亚系CEM / R30dm通过反复短期（24小时）的CEM-WT细胞暴露于30μM的MTX作为模拟的临床方案开发的。有趣的是，该细胞系也显示塞米松交叉耐药（未发表的结果）的量，机制尚不完全清楚。 固体TUM或模型在本研究调查的是胰腺导管腺癌，臭名昭著的非凡耐火度化疗。为此，我们选择的Panc-1细胞系和由连续孵育与药物24的1μM得到其耐吉西他子克隆的Panc-1R。在这里，我们描述了一种方法通过组合三个协议来发现在体外耐药底层新颖机制:比色细胞毒性试验，以评估在白血病细胞和实体瘤，RNA-SEQ基于管道癌细胞药物敏感性，以确定相关的新的剪接变体药物敏感性/抗性和RT-PCR和定量RT-PCR分析，以验证潜在候选。

<table><tbody><tr><td>Sulforhodamine B</td><td>Sigma-Aldrich</td><td>230162</td><td></td></tr><tr><td>Trichloroacetic acid</td><td>Sigma-Aldrich</td><td>251399</td><td></td></tr><tr><td>CCRF-CEM</td><td>ATCC, Manassas, VA, USA</td><td>ATCC CCL-119</td><td></td></tr><tr><td>Panc-1</td><td>ATCC, Manassas, VA, USA</td><td>ATCC CRL-1469</td><td></td></tr><tr><td>DMEM high glucose</td><td>Lonza, Basel, Switzerland</td><td>12-604F</td><td></td></tr><tr><td>RPMI-1640&amp;nbsp;</td><td>Gibco, Carlsbad, CA, USA</td><td>11875093</td><td></td></tr><tr><td>Fetal bovine and calf serum</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>758093</td><td></td></tr><tr><td>penicillin G streptomycin sulphate</td><td>Gibco, Carlsbad, CA, USA</td><td>15140122</td><td></td></tr><tr><td>Tris(hydroxymethyl)-aminomethane</td><td>Sigma Aldrich</td><td>252859</td><td></td></tr><tr><td>MTT formazan</td><td>Sigma Aldrich</td><td>M2003</td><td></td></tr><tr><td>Anthos-Elisa-reader 2001</td><td>Labtec, Heerhugowaard, Netherlands</td><td></td><td>UV-Vis 96-well plate spectrophotometer</td></tr><tr><td>Greiner CELLSTAR 96 well plates</td><td>Greiner/Sigma</td><td>M0812-100EA</td><td></td></tr><tr><td>Trypsin/EDTA Solution 100 ml</td><td>Lonza, Basel, Switzerland</td><td>CC-5012</td><td></td></tr><tr><td>CELLSTAR Cell Culture Flasks 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>82051-074</td><td></td></tr><tr><td>CELLSTAR Cell Culture Flasks 75 cm&lt;sup&gt;3&lt;/sup&gt;</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>82050-856</td><td></td></tr><tr><td>Phosphate Buffered Saline (NaCl 0.9%)</td><td>B.Braun Melsungen AG, Germany</td><td>362 3140</td><td></td></tr></tbody></table>

using rna-sequencing to detect novel splice variants related to drug resistance in in vitro cancer models

抗药性仍然是癌症治疗为恶性血液病和实体瘤的一个主要问题。固有或获得性可通过多种机制，包括药物的消除增加引起的，药物吸收，药物失活和药物靶点的改变降低。最近的数据表明，除了通过公知的基因（突变，扩增）和表观遗传（DNA的甲基化，组蛋白的翻译后修饰）的修改，耐药机制也可能会被拼接像差调节。这是调查一个值得今后注意，以规划更加有效的治疗方法的迅速增长领域。在本文中描述的协议的目的是调查异常剪接的耐药性在实体瘤和恶性血液病的影响。为了这个目标，我们通过RNA测序和分析建立在体外模型几个转录概况ED一个定量RT-PCR为基础的方法来验证候选基因。特别是，我们评估DDX5和PKM转录物的差别剪接。由计算工具MATS检测出的异常剪接在白血病细胞进行了验证，显示出不同DDX5剪接变体是在亲代与抗性细胞中表达。在这些细胞中，我们也观察到了更高PKM2 / PKM1比，这未在的Panc-1抗吉西他滨的对应检测相比亲Panc-1细胞，这表明由吉西他滨诱导的曝光耐药的不同的机制。

在协议中所描述的细胞毒性测定法提供了可靠的和可靠的方法对癌细胞的电阻评估体外化疗剂。 CEM / R30dm，CEM-R5和CEM-C3:通过MTT测定法的手段，以地塞米松灵敏度在四个T-ALL细胞系，包括地塞米松敏感亲代的CEM-WT细胞，和三个塞米松抗性亚系进行了测定。两种不同的浓度范围不得不由于用来在灵敏度CEM-WT之间（2μM - 0.97纳米）的大的差异和地塞米松抗性细胞系（640μM ...

Watch this Scientific Journal Video about Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models at JoVE.com

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

这里，我们描述旨在调查异常剪接的耐药性在实体瘤和血液恶性肿瘤的影响的协议。为了这个目标，我们通过RNA测序分析，家长和体外抗车型的转录配置文件，并建立了定量RT-PCR为基础的方法来验证候选基因。

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 ...

using-rna-sequencing-to-detect-novel-splice-variants-related-to-drug

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

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

13.6K 次观看.  VU University Medical Center. 这里，我们描述旨在调查异常剪接的耐药性在实体瘤和血液恶性肿瘤的影响的协议。为了这个目标，我们通过RNA测序分析，家长和体外抗车型的转录配置文件，并建立了定量RT-PCR为基础的方法来验证候选基因。 

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

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

科研

遗传学

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

利用下一代测序检测 CtDNA 中的稀有突变

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

癌症研究

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

有针对性的下一代测序和生物信息学管道评价宪政疾病的遗传决定因素

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

使用 E1A 微型工具研究 mRNA 拼接更改

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

<meta charset="utf8"></head><body>使用野生型封闭 PCR 优化低频突变检测

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.

<meta charset="utf8"></head><body>作者聚焦：推进癌症组织中低频突变的检测

This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other diseases.

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

生物学

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

一种基于报告基因的细胞检测，用于监测剪接效率

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

生物化学

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

实施<em&gt;体外</em&gt;耐药性检测:最大化的潜力，揭开临床有关的耐药机制

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

利用RNA测序来检测新剪接变异体在相关耐药

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合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

利用下一代测序检测 CtDNA 中的稀有突变

有针对性的下一代测序和生物信息学管道评价宪政疾病的遗传决定因素

使用 E1A 微型工具研究 mRNA 拼接更改

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

一种基于报告基因的细胞检测，用于监测剪接效率

实施