作者要感谢健康NHLBI的计划项目资助的国家研究院的嗜酸性粒细胞气道炎症和重塑的作用:P01HL088584（PI:N. Jarjour）和威斯康星州Carbone的癌症中心和医学系的大学对于校内资助。我们感谢道格拉斯·安妮斯克隆四个STAT3变种。

注:未识别信息在符合由威斯康星 - 麦迪逊中心大学健康科学机构审查委员会的协议获得批准（＃1513至70年）共收到外周血嗜酸性粒细胞。对于使用在研究各试样的获得从供体签署了知情同意书。 1.创建质粒为模板的标准<ol><li>对于扩增子跨越两个STAT3剪接位点创造的引物，用5&#39;-KpnI和NheI位限制性位点（5&#39;-扩展）按表1a。 注:KpnI和NheI位被选择，以使插入到被连接到用卡那霉素抗性25,26的变形的pET-28a中的矢量KpnI位点已被加入到多克隆位点。 </li><li>使用新鲜捐赠嗜酸性粒细胞，在细胞培养基中制备的2.5×10 6个细胞/ ml的两份样品（罗斯韦尔园区MEMORIA升研究所（RPMI）1640培养基）。在37℃下，5％CO 2和10％湿度下孵育1小时。 <ol><li>从样品制备互补（三）的DNA（每个制剂在至少1×10 6个细胞），为每使用RNA提取和cDNA合成试剂盒制造商的说明。 </li></ol></li><li>从步骤1.2.1编写模板的cDNA，扩增STAT3剪接变异体用PCR扩增按表2a。 </li><li>解决在2％琼脂糖Tris-乙酸盐-乙二胺（TAE）凝胶27的扩增子。从凝胶消费频带和根据凝胶切除试剂盒的说明纯化。 </li><li>切割扩增子和质粒用适当的限制性内切酶（在参考27限制的概述），采用卷，孵育时间和温度推荐酶提供商;和纯化如在步骤1.4。结扎供应商推荐的条件下，限制扩增到质粒。 </li><li>准备一个负续列伊（限制质粒DNA的插入，而不但有连接酶）和阳性对照（含卡那霉素抗性质粒不受限制）。执行主管E.标准质粒转化大肠杆菌 DH5α28使用连接混合物27。 </li><li>孵育转化E.大肠杆菌在1ml的Luria肉汤（LB）培养基（如指示27制备的）在37℃振荡1小时。在含有50μg/ ml卡那霉素，并在37℃下孵育过夜的LB平板上传播转化体。 </li><li>使用无菌牙签27 STAT3α和βSTAT3板块选择几个克隆。每次转移〜2毫升LB.生长培养过夜，在37℃在振荡培养箱中。 </li><li>通过以下中的DNA纯化试剂盒提供的指令净化从细菌培养物的质粒。商店质粒在-20℃或-80℃。 </li><li>通过准备20微升测序反应从几个克隆序列质粒。移液器3微升测序反应缓冲液，2微升测序反应混合物，12微升的超纯水，1μl的质粒作为模板和2μl测序引物。 注意:如果使用PET-28A载体，用测序引物5&#39;-TAA TAC GAC TCA CTA TAG GGG-3&#39;。 <ol><li>编码评估每个变体的质粒electrophoretograms:Sα，Sβ，ΔSα和ΔSβ25。 </li></ol></li><li>在毫微克/微升纯质粒DNA的浓度测量。如果使用分光光度计在260nm读取吸光度，使用比尔-朗伯定律29计算DNA浓度: <img alt="式（1）" src="/files/ftp_upload/54473/54473eq1.jpg" /> 其中c =浓度，A =吸光度，ε（消光系数）= 0.02（微克/毫升）-1厘米-1的光的双链DNA和L =通路长度（通常为1厘米）。 </li><li>用STAT3剪接变体的cDNA点的分子量plicons和向量，计算出每微升的拷贝数。 注意:每个碱基对分子量（bp）的被估计为650道尔顿。 <img alt="公式（2）" src="/files/ftp_upload/54473/54473eq2.jpg" /></li></ol> 2.分析引物特异性绝对定量PCR <ol><li>在10稀释系列的STAT3的质粒的制备1，用超纯水每微升〜 八月 10日至3月10日份（20微升）。最集中的测量浓度（每微升10〜8份，见步骤1.11）。 </li><li>用稀释质粒准备四"非目标"混合物（ 即含STAT3Sα，ΔSα，Sβ但没有ΔSβ的浓度相等STAT3ΔSβ阴性对照），每微升每10 6拷贝。每微升10 6拷贝，准备以同样的浓度每四个模板的"目标"组合。 </li><li>准备引物对解决方案是对每一个剪接变体（ 参见表1b和图1）通过使〜60微升的每个引物在7μM浓度（在样品的最终浓度将是560纳米）。 </li><li>稀释DNA聚合酶/双链DNA结合染料混合7:5的纯H 2 O. </li><li>如图模板（ 表3）成立试验1在96孔PCR板。 <ol><li>加入21微升稀释DNA聚合酶/双链DNA结合染料混合，然后2微升引物混合和2微升模板（按表2b）。相反模板，加入2微升过滤H 2 O操作无模板对照（NTC）好。双份的反应来评估可重复性30。 </li></ol></li><li>密封的qPCR板粘接盖，离心在1200 XG在12℃5分钟。 </li><li>如果使用的材料上市的软件，如描述设置实验。 <ol><li>打开定量PCR机上插入密封定量PCR板。点击文件＆＃62;新建&gt;下一页&gt;选择"新建器"，选择记者，淬火并提供名称。设置为每个剪接变体"新探测器"。 </li><li>选择下一步，设置为提示设置试板网页标准，确保数量输入到表和相应的探测器选择。单击Finish。 </li><li>设置循环按表2b。确保荧光读数（以确定的Ct）在每个周期的72℃步骤发生。选择运行。 </li><li>当运行完成后，单击结果选项卡&gt;扩增图标签。评估输出扩增曲线，以评估数据质量（找一个指数的情节， 如图2）。 注:确保扩增曲线大多是指数和周期的阈值可以在情节的指数部分进行设置。确保NTC却没有放大和其他标准示范点，具有高ΔRn低Ct值。 </li><li>与EXPORT结果到电子表格软件，点击文件&gt;导出&gt;结果。 </li></ol></li></ol> 3.评估绝对qPCR分析特异性和重复性<ol><li>重复性<ol><li>使用来自步骤2.7.5的数据来计算的Ct标准偏差值（荧光阈值循环）。 <img alt="公式3" src="/files/ftp_upload/54473/54473eq3.jpg" /> 其中N =（2如果一式两份进行）的样本数， <img alt="公式4" src="/files/ftp_upload/54473/54473eq4.jpg" /> =样品的CT和<img alt="公式5" src="/files/ftp_upload/54473/54473eq5.jpg" /> =样品的Ct的意思。检查重复的Ct值的标准偏差≤0.2。检查，但在所有NTC井Ct值比38小。 </li><li>如果重复性通过增加或减少退火时间差优化循环参数。 </li></ol></li><li>情节日志副本麻木器（如在步骤1.12确定，并且在步骤2.1制得）与Ct值 创建标准曲线;得到以下公式: <img alt="公式6" src="/files/ftp_upload/54473/54473eq6.jpg" /> 其中y是CT，m是梯度，x是日志（拷贝数），而b是截距值。 注:线性回归的R 2值将是≥0.95为良好的数据。 </li><li>使用标准曲线和方程计算扩增效率: <img alt="公式7" src="/files/ftp_upload/54473/54473eq7.jpg" /> 注:瞄准效率≥75％。 </li><li>使用太多的公式由qPCR分析1计算特异性: <img alt="式（8）" src="/files/ftp_upload/54473/54473eq8.jpg" /> 其中Δ 的Ct 太 = 的Ct 目标 - 的Ct 非目标 ，描述用于特异性和非特异性放大器在阈值循环数之差lification 注:特异性应该超过四个数量级（ 即 ，特异性因子≥4）。 <ol><li>如果特异性较差，通过降低引物浓度优化。设置测定用最终的引物浓度范围为100至500nM（如步骤2.5-2.7中描述）。 </li></ol></li></ol> 4.执行相对定量PCR检测<ol><li>处理细胞与细胞因子促进转录的。 <ol><li>对于新鲜捐赠嗜酸性粒细胞，制备的在细胞培养基中2.5×10 6个细胞/ ml（RPMI 1640培养基）两份样品，并用50纳克/毫升白细胞介素3（IL3）和/或50纳克/毫升肿瘤坏死因子α治疗（ TNFα）在37℃，5％CO 2和10％湿度下3，6,9和20小时的周期。 </li></ol></li><li>从嗜酸性粒细胞的样品（每个制剂在至少1×10 6个细胞），为每使用RNA提取和cDNA合成试剂盒制造商的说明制备cDNA。 </li&gt; <li>准备两个样品的cDNA等分试样（对照或静止样本）的连续稀释液，以从"1"的稀释度范围为"1 1024"稀释。 <ol><li>为1/4的稀释，移液管将1μlcDNA和3微升超纯水。 </li><li>对于1 16稀释，移液管1的1微升4稀释和3微升超纯水等 。 </li></ol></li><li>设置的PCR在96孔板用25微升的反应所描述的步骤，2.3-2.5但具有几个引物浓度为泛STAT3和GUSB（ 见表4模板）。 </li><li>添加"新的探测器"为GUSB并按照步骤2.6-2.7，利用表2c描述的循环参数。 </li><li>生成标准曲线（见步骤3.2和3.3），以确定哪些引物浓度导致95％-100％的扩增效率。 </li><li>设置板的cDNA样品（步骤4.2）和优化的引物浓度（参见循环参数，试剂和表5的96孔板模板表2c）。 </li><li>执行步骤2.6-2.7。重复测定至少一次，检查数据质量。 </li><li>计算为STAT3和看家基因GUSB重复样本CT的平均Ct值。 </li><li>获得用于每个样品ΔCt值。 <img alt="公式9" src="/files/ftp_upload/54473/54473eq9.jpg" /></li><li>指定的休息样本（通常有兴趣成绩单的最低浓度）样品0计算ΔΔ31 的Ct每个样品（这里指定为样本X）: <img alt="10式" src="/files/ftp_upload/54473/54473eq10.jpg" /></li><li>计算倍数增加每个样品: <img alt="11式" src="/files/ftp_upload/54473/54473eq11.jpg" /></li><li>再生性<ol><li>计算偏差（CV）的拷贝数泛ST系数AT3和GUSB。 <img alt="12式" src="/files/ftp_upload/54473/54473eq12.jpg" /> 其中n =样本数，=采样的计算拷贝数和=样本均值。检查每个样品（多种分析）的那CV值≤10％。 </li></ol></li></ol> 5.分析绝对定量PCR数据未知样品<ol><li>比较相对定量PCR获得（如步骤4.9所述）的Ct值看家基因GUSB，以确保样本&#39;的cDNA浓度为一个数量级之内。 <ol><li>稀释的cDNA相应（ 例如，如果样品1具有的Ct值〜17.5，与其它样品" 的Ct值是〜21，样品1是大致2（21-17.5）= 11倍浓缩执行1:1稀释相同的范围内的超纯水，而不超过必要稀释以上）。 注:迥然不同的起始浓度将偏置线性回归分析（步4.12）。 </li></ol></li><li>设置样本的绝对qPCR分析。准备试验2A模板板Sα和Sβ按表6;并准备ΔSα和ΔSβ测定2b中按表7中 。开展试验如在步骤中描述2.6-2.7.3（和表2b）。重复测定至少一次。 </li><li>检查数据质量和出口为一体的步骤2.7.4-2.7.5描述。 </li><li>设置绝对定量PCR的96孔板用泛STAT3引物（在表1c的引物，在表8的模板），在400μM和所有四种质粒的混合物或在列出总浓度的单一质粒25μl的反应。 注:效率不要紧绝对定量PCR，但优化这些引物效率是用于相对定量PCR（步骤4）重要。 </li><li>密封的qPCR板粘接盖，离心在1200 XG在12℃5分钟。 </li><li>成立了"新的探测器"泛- STAT3如步骤2.7.1-2.7.3。 </li><li>设置为循环STAT3泛按表2c（相同条件相对定量PCR）。确保荧光读数（以确定的Ct）在每个周期的72℃步骤发生。重复测定至少一次，以确保重现性。 </li><li>检查数据质量和出口（见步骤2.7.4-2.7.5）。 <ol><li>如果扩增曲线不是指数（参见图2），稀样品的cDNA（1:10）和描述（步骤5.7）重复测定。 </li></ol></li><li>利用标准曲线的方程（参见3.2， 图3）和样品的Ct值，计算每个剪接变体，并且每个样品中泛STAT3的拷贝数。 <img alt="13式" src="/files/ftp_upload/54473/54473eq13.jpg" /></li><li>地块的日志日志VS（累计绝对定量PCR拷贝数STA值（泛STAT3获得绝对定量PCR拷贝数的值）T3剪接变异体）。 注:理想的线性回归和斜率都希望能〜1。 </li><li>计算可重复性（步骤3.1）和可重复性（步骤4.13）。 </li></ol> 6.合并绝对和相对定量PCR数据<ol><li>乘变体与总STAT3倍数增加来计算每个剪接变体的倍数增加的比例。 </li><li>计算的标准偏差（见步骤3.1.1）的绝对值和相对值的占错误的传播。确定测量的标准误差（SEM），如下所示: <img alt="14式" src="/files/ftp_upload/54473/54473eq14.jpg" /></li></ol>

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	<li>Hiller, 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=Phylogenetically+widespread+alternative+splicing+at+unusual+GYNGYN+donors.">Phylogenetically widespread alternative splicing at unusual GYNGYN donors.</a> Genome Biol. 7 (7), R65 (2006).</li><li>Treacy, M. 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=Twin+of+I-POU:+a+two+amino+acid+difference+in+the+I-POU+homeodomain+distinguishes+an+activator+from+an+inhibitor+of+transcription.">Twin of I-POU: a two amino acid difference in the I-POU homeodomain distinguishes an activator from an inhibitor of transcription.</a> Cell. 68 (3), 491-505 (1992).</li><li>Schindler, 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=Alternative+splicing+at+NAGNAG+acceptors+in+Arabidopsis+thaliana+SR+and+SR-related+protein-coding+genes.">Alternative splicing at NAGNAG acceptors in Arabidopsis thaliana SR and SR-related protein-coding genes.</a> BMC Genomics. 9, 159 (2008).</li><li>Szafranski, K., Kramer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=It's+a+bit+over,+is+that+ok?+The+subtle+surplus+from+tandem+alternative+splicing.">It's a bit over, is that ok? The subtle surplus from tandem alternative splicing.</a> RNA Biol. 12 (2), 115-122 (2015).</li><li>Wang, 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=Alternative+splicing+at+GYNNGY+5'+splice+sites:+more+noise,+less+regulation.">Alternative splicing at GYNNGY 5' splice sites: more noise, less regulation.</a> Nucleic Acids Res. 42 (22), 13969-13980 (2014).</li><li>Hiller, M., Platzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+and+subtle:+alternative+splicing+at+short-distance+tandem+sites.">Widespread and subtle: alternative splicing at short-distance tandem sites.</a> Trends Genet. 24 (5), 246-255 (2008).</li><li>Tsai, K. W., Lin, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+wobble+splicing+indicates+that+it+is+not+tissue+specific.">Quantitative analysis of wobble splicing indicates that it is not tissue specific.</a> Genomics. 88 (6), 855-864 (2006).</li><li>Tadokoro, K., 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=Frequent+occurrence+of+protein+isoforms+with+or+without+a+single+amino+acid+residue+by+subtle+alternative+splicing:+the+case+of+Gln+in+DRPLA+affects+subcellular+localization+of+the+products.">Frequent occurrence of protein isoforms with or without a single amino acid residue by subtle alternative splicing: the case of Gln in DRPLA affects subcellular localization of the products.</a> J Hum Genet. 50 (8), 382-394 (2005).</li><li>Bradley, R. K., Merkin, J., Lambert, N. J., Burge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+RNA+triplets+is+often+regulated+and+accelerates+proteome+evolution.">Alternative splicing of RNA triplets is often regulated and accelerates proteome evolution.</a> PLoS Biol. 10 (1), e1001229 (2012).</li><li>Zheng, C. L., Fu, X. D., Gribskov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+regulatory+elements+defining+constitutive+splicing+and+different+modes+of+alternative+splicing+in+human+and+mouse.">Characteristics and regulatory elements defining constitutive splicing and different modes of alternative splicing in human and mouse.</a> RNA. 11 (12), 1777-1787 (2005).</li><li>Schaefer, T. S., Sanders, L. K., Nathans, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+transcriptional+activity+of+Jun+and+Stat3+beta,+a+short+form+of+Stat3.">Cooperative transcriptional activity of Jun and Stat3 beta, a short form of Stat3.</a> Proc Natl Acad Sci U S A. 92 (20), 9097-9101 (1995).</li><li>Waitkus, M. 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=Signal+integration+and+gene+induction+by+a+functionally+distinct+STAT3+phosphoform.">Signal integration and gene induction by a functionally distinct STAT3 phosphoform.</a> Mol Cell Biol. 34 (10), 1800-1811 (2014).</li><li>Srivastava, J., DiGiovanni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-canonical+Stat3+signaling+in+cancer.">Non-canonical Stat3 signaling in cancer.</a> Mol Carcinog. , (2015).</li><li>Holland, S. 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=STAT3+mutations+in+the+hyper-IgE+syndrome.">STAT3 mutations in the hyper-IgE syndrome.</a> N Engl J Med. 357 (16), 1608-1619 (2007).</li><li>Maritano, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STAT3+isoforms+alpha+and+beta+have+unique+and+specific+functions.">The STAT3 isoforms alpha and beta have unique and specific functions.</a> Nat Immunol. 5 (4), 401-409 (2004).</li><li>Hevehan, D. L., Miller, W. M., Papoutsakis, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+and+phosphorylation+of+distinct+STAT3+proteins+during+granulocytic+differentiation.">Differential expression and phosphorylation of distinct STAT3 proteins during granulocytic differentiation.</a> Blood. 99 (5), 1627-1637 (2002).</li><li>Yoo, J. Y., Huso, D. L., Nathans, D., Desiderio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+ablation+of+Stat3beta+distorts+the+pattern+of+Stat3-responsive+gene+expression+and+impairs+recovery+from+endotoxic+shock.">Specific ablation of Stat3beta distorts the pattern of Stat3-responsive gene expression and impairs recovery from endotoxic shock.</a> Cell. 108 (3), 331-344 (2002).</li><li>Stahl, 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=Choice+of+STATs+and+other+substrates+specified+by+modular+tyrosine-based+motifs+in+cytokine+receptors.">Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors.</a> Science. 267 (5202), 1349-1353 (1995).</li><li>Zheng, 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=A+mix+of+S+and+DeltaS+variants+of+STAT3+enable+survival+of+activated+B-cell-like+diffuse+large+B-cell+lymphoma+cells+in+culture.">A mix of S and DeltaS variants of STAT3 enable survival of activated B-cell-like diffuse large B-cell lymphoma cells in culture.</a> Oncogenesis. 4, 184 (2016).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Zipper, H., Brunner, H., Bernhagen, J., Vitzthum, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigations+on+DNA+intercalation+and+surface+binding+by+SYBR+Green+I,+its+structure+determination+and+methodological+implications.">Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications.</a> Nucleic Acids Res. 32 (12), e103 (2004).</li><li>Kelly, E. A., Liu, L. Y., Esnault, S., Quinchia Johnson, B. H., Jarjour, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+synergistic+effect+of+IL-3+and+TNF+on+matrix+metalloproteinase+9+generation+by+human+eosinophils.">Potent synergistic effect of IL-3 and TNF on matrix metalloproteinase 9 generation by human eosinophils.</a> Cytokine. 58 (2), 199-206 (2012).</li><li>Whelan, J. A., Russell, N. B., Whelan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+absolute+quantification+of+cDNA+using+real-time+PCR.">A method for the absolute quantification of cDNA using real-time PCR.</a> J Immunol Methods. 278 (1-2), 261-269 (2003).</li><li>Too, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+PCR+quantification+of+GFRalpha-2+alternatively+spliced+isoforms+in+murine+brain+and+peripheral+tissues.">Real time PCR quantification of GFRalpha-2 alternatively spliced isoforms in murine brain and peripheral tissues.</a> Brain Res Mol Brain Res. 114 (2), 146-153 (2003).</li><li>Turton, K. B., Annis, D. S., Rui, L., Esnault, S., Mosher, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratios+of+Four+STAT3+Splice+Variants+in+Human+Eosinophils+and+Diffuse+Large+B+Cell+Lymphoma+Cells.">Ratios of Four STAT3 Splice Variants in Human Eosinophils and Diffuse Large B Cell Lymphoma Cells.</a> PloS One. 10 (5), e0127243 (2015).</li><li>Maurer, 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=Extended+binding+site+on+fibronectin+for+the+functional+upstream+domain+of+protein+F1+of+Streptococcus+pyogenes.">Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes.</a> J Biol Chem. 285 (52), 41087-41099 (2010).</li><li>Ausubel, F. 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=.">.</a> Current protocols in molecular biology. , (1987).</li><li>Grant, S. G., Jessee, J., Bloom, F. R., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+plasmid+rescue+from+transgenic+mouse+DNAs+into+Escherichia+coli+methylation-restriction+mutants.">Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants.</a> Proc Natl Acad Sci U S A. 87 (12), 4645-4649 (1990).</li><li>Kocsis, L., Herman, P., Eke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+modified+Beer-Lambert+law+revisited.">The modified Beer-Lambert law revisited.</a> Phys Med Biol. 51 (5), N91-N98 (2006).</li><li>Bustin, S. A., 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+MIQE+guidelines:+minimum+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.</a> Clin Chem. 55 (4), 611-622 (2009).</li><li>Pfaffl, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mathematical+model+for+relative+quantification+in+real-time+RT-PCR.">A new mathematical model for relative quantification in real-time RT-PCR.</a> Nucleic Acids Res. 29 (9), e45 (2001).</li><li>Bustin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A-Z of quantitative PCR. , (2004).</li><li>Zhang, H. F., Lai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+in+cancer-friend+or+foe.">STAT3 in cancer-friend or foe.</a> Cancers (Basel). 6 (3), 1408-1440 (2014).</li><li>Caldenhoven, E., 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=STAT3beta,+a+splice+variant+of+transcription+factor+STAT3,+is+a+dominant+negative+regulator+of+transcription.">STAT3beta, a splice variant of transcription factor STAT3, is a dominant negative regulator of transcription.</a> J Biol Chem. 271 (22), 13221-13227 (1996).</li><li>Zammarchi, F., 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=Antitumorigenic+potential+of+STAT3+alternative+splicing+modulation.">Antitumorigenic potential of STAT3 alternative splicing modulation.</a> Proc Natl Acad Sci U S A. 108 (43), 17779-17784 (2011).</li><li>Liu, Y., Beyer, A., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Dependency+of+Cellular+Protein+Levels+on+mRNA+Abundance.">On the Dependency of Cellular Protein Levels on mRNA Abundance.</a> Cell. 165 (3), 535-550 (2016).</li><li>Narimatsu, 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=Tissue-specific+autoregulation+of+the+stat3+gene+and+its+role+in+interleukin-6-induced+survival+signals+in+T+cells.">Tissue-specific autoregulation of the stat3 gene and its role in interleukin-6-induced survival signals in T cells.</a> Mol Cell Biol. 21 (19), 6615-6625 (2001).</li><li>Szafranski, K., 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=Physiological+state+co-regulates+thousands+of+mammalian+mRNA+splicing+events+at+tandem+splice+sites+and+alternative+exons.">Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons.</a> Nucleic Acids Res. 42 (14), 8895-8904 (2014).</li><li>Sundin, 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=Novel+STAT3+mutation+causing+hyper-IgE+syndrome:+studies+of+the+clinical+course+and+immunopathology.">Novel STAT3 mutation causing hyper-IgE syndrome: studies of the clinical course and immunopathology.</a> J Clin Immunol. 34 (4), 469-477 (2014).</li><li>Oltean, S., Bates, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+alternative+splicing+in+cancer.">Hallmarks of alternative splicing in cancer.</a> Oncogene. 33 (46), 5311-5318 (2014).</li><li>Boultwood, J., Dolatshad, H., Varanasi, S. S., Yip, B. H., Pellagatti, A. <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+splicing+factor+mutations+in+the+pathogenesis+of+the+myelodysplastic+syndromes.">The role of splicing factor mutations in the pathogenesis of the myelodysplastic syndromes.</a> Adv Biol Regul. 54, 153-161 (2014).</li><li>Tilgner, 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=Comprehensive+transcriptome+analysis+using+synthetic+long-read+sequencing+reveals+molecular+co-association+of+distant+splicing+events.">Comprehensive transcriptome analysis using synthetic long-read sequencing reveals molecular co-association of distant splicing events.</a> Nat Biotechnol. 33 (7), 736-742 (2015).</li><li>Hiller, 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=Widespread+occurrence+of+alternative+splicing+at+NAGNAG+acceptors+contributes+to+proteome+plasticity.">Widespread occurrence of alternative splicing at NAGNAG acceptors contributes to proteome plasticity.</a> Nat Genet. 36 (12), 1255-1257 (2004).</li><li>Rosenfeld, J. A., Malhotra, A. K., Lencz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+multi-nucleotide+polymorphisms+in+the+human+genome+characterized+by+whole+genome+and+exome+sequencing.">Novel multi-nucleotide polymorphisms in the human genome characterized by whole genome and exome sequencing.</a> Nucleic Acids Res. 38 (18), 6102-6111 (2010).</li></ol>

The authors have nothing to disclose.

我们以评估在嗜酸性粒细胞和淋巴瘤细胞的水平和STAT3剪接变体转录物的比例和学习细胞因子刺激是否影响了水平和比例开发了这个协议。 STAT3是由于其多效性的和不确定的功能特别感兴趣的，具有相互冲突是否它作为在癌症的癌蛋白或肿瘤抑制的报告（参照33中综述）。在STAT3α和β剪接变异体的功能差异已被定性之前34,35，和我们的协议促进了击倒/重新表达分析表明，这需要所组成的最佳比例和ΔS成绩单19。 不同的剪接变异体的精确定量将促进有关异构STAT3功能剪接变异体组成的进一步调查。协议集成绝对和相对的qPCR数据，结合Ab的能力溶质的qPCR来计算剪接变体的比例，及相对的qPCR来测量总STAT3的表达的变化。这种方法允许人们在两个备选剪接位点相距多于50个核苷酸区分在序列的细微差别，并同时测量剪接比率。单独确定剪接事件的比例就不会产生一个共同的拼接偏压存在使得ΔSβ水平比如果两个站点的使用是随机剪接25预期更高的显着的发现。 重要的是，绝对定量PCR与使用质粒校准曲线使量化（在次最佳效率）可产生高度相似的序列拼接的变化。我们期待一个新的微妙拼接qPCR分析应该采取大约两个月的时间来进行优化。在检测开发的关键步骤是生成绝对定量PCR标准曲线用于STAT3质粒的建立;实验确定最佳的引物序列和循环参数，以确保特异性和重复性;和相对定量PCR数据的整合，从相对于GAPDH表达泛STAT3的表达量化的。拷贝数由泛STAT3与定量累计定量的相关性（Sα+ΔSα+Sβ+ΔSβ）表明，该协议产生可靠的结果。 该技术的需要注意的是广泛的验证过程。有必要评估测定内变异（重复性），批间变异性（再现性）和特异性。该协议概括的方式来获得这些参数的数字输出。我们认为效率≥75％，特异性因素≥4，变异（重复性）的系数≤10％及的Ct标准偏差（重复性）≤0.2为宜阈值30。在STAT3序列的氨基酸1-690突变或缺失不会discove通过该协议的红色，虽然他们可能会影响拼接比率。转录比例可能不成正比proteoform比例36。 由于样本有不同的总cDNA的起始量，绝对定量PCR适合使用，除非已建立的看家基因加上相对定量PCR样品中比较剪接变异体的拷贝数，但不适用于样本间的比较。该方法描述符合MIQE定量PCR准则重复性30。 PCR循环参数和引物的浓度可能需要进行修改，如果使用其它设备，以获得可再现的数据。完美的特异性不可能的，而不显着损害效率，但目标是由大于四量级的更有效的扩增。 线性DNA更容易不是圆形扩增。如果备用的质粒不能提供满意的标准曲线（R 2 &lt;; 0.95），考虑之前的量化线性单网站限制的质粒。优化的qPCR为获得良好的质量的数据（ 图1）是至关重要的。大多数的qPCR协议依赖于两个步骤的循环，和机器也相应进行了优化。加热块的非均匀加热可以在三步骤循环而加剧，有助于重复性差。试验必须带有过滤器的吸头和超纯水在无菌条件下成立，最好是在一个专用的层流罩。因为污染物可导致不一致的结果，测定应在有滤波器枪头和超纯水在无菌条件下成立，最好在专用层流罩。有关定量PCR优化的详细信息，请参阅巴斯廷等 32 量化STAT3可能导致一些环境的更深入的了解。 STAT3自动调节自身的表达37，和上述的协议可帮助阐明STAT3剪接变异体的比例是否有助于调节这一正反馈环。该协议可以被用作在不同密度38或以上的发展过程中细胞中观察到研究剪接变体的比率的变化:已知的是在造血16中蛋白质水平的STAT3α/β比的变化。 Sundin的等 。发现12号外显子的STAT3内含子单核苷酸多态性偏见拼接与工作综合征39患者。可以想象的是，存在于外显子21和22，或外显子22和23之间的内含子的许多个SNP中的一个可以分别向ΔS/ S的剪接比率和α/β。该测定可用于量化在癌细胞，其中突变或改变在剪接调控可能引入偏倚的剪接过程40 的STAT3转录。在剪接因子突变（象SF3B1），如OBSERVED骨髓增生异常综合征41也可能导致可通过该协议来衡量变化。 更广泛地说，该方法特异性检测共协会在剪接，这是不符合常规RNA测序，也不标准定量PCR可行。而相互排斥的外显子剪接的现象证明了的拼接决定协调，其他剪接事件的联合协会尚未精心研究。一个最近所描述的替代方法，其中，RNA测序被修改，以便询问全长cDNA，表明遥远剪接事件是多种共依赖性比以前认为的42。 STAT3包含了供体串联剪接位点。受体串联剪接位点是更频繁的43和概述协议的原理可以作为一个起点，显影测定法重合检测NAGNAG剪接和内200个核苷酸的其他剪接事件。其他POTENTiAl基合金的应用包括其他细微序列差异，如插入缺失或双/三核苷酸多态性44重合的定量。

短程（串联）的选择性剪接，其中备用受体或供体位点是靠近彼此，是在哺乳动物中1，无脊椎动物2和植物3常见。据估计，哺乳动物基因的20％含有替代剪接位点2-12个核苷酸隔开4。许多站点的相隔3个核苷酸，并导致在单个密码子的包括或排除。有一个在这些网站5,6一些人认为辩论有关拼接法规性质的拼接图案的差异是如此微妙的选择是随机的7，而另一些基于组织特异性8推断监管。 串联剪接位点的选择已经分析半定量使用改性毛细管电泳7，和高分辨率凝胶电泳8。 RNA测序（RNA测序）读取可用于在每个接合量化拼接比率现场。以这种方式，RNA测序数据已经提供的洞察的串联剪接位点9的调节。它还使基于核苷酸序预计10剪接变异率的预测。在大多数拼接的重点，其中包括或排除一个密码子已经上更经常发生的串联受体剪接位点，被称为NAGNAGs（其中n =任何核苷酸）的。 串联捐助替代剪接位点包括或不包括单一密码子（GYNGYN识别基序，其中Y =嘧啶）比串联受体较少见。信号转导和转录3（STAT3）的激活剂是经历串联捐助剪接1,11的一个关键基因。串联供体剪接位点加入外显子21和22，并导致在密码子的包括或排除对丝氨酸-701（S或ΔS分别）1,11。下游替代受体位点（40个核苷酸彼此分开）接合的外显子22和23A / B结果在列入激活结构域（α）或具有7独特的C端残基（β）11截短激活域的任一55末端残基的。因此，四剪接变体是可能的。 STAT3蛋白在多种细胞类型12的转录因子和主要信号集成商和突变时，其构激活有助于癌症的几个表型（参考文献13综述）。工作综合征，免疫缺陷疾病的特点是高水平的IgE，也引起了STAT3基因突变（参照14综述）。对于STAT3α和β剪接变体蛋白不同的角色已经先前所述15。最初，STAT3β被认为在一个显性负方式16行事，拮抗STAT3α的转录活性，但随后的工作表明它具有独立的靶基因17 </s了&gt;。尽管串联拼接的精妙之处，有理由相信没有或丝氨酸701（Ser701）影响功能的存在。不仅是Ser701靠近酪氨酸-705（残余物中STAT3的活化18磷酸化），但最近的研究表明，STAT3 S和ΔS剪接变体都必需STAT3-嗜弥漫大B细胞淋巴瘤（DLBLCL）的活力细胞19。生物相关性还有待探索。鉴于剪接变体组合物可以影响的功能，我们努力发现是否比通过细胞因子的刺激在嗜酸性粒细胞扰动。 起初，我们试图通过使用PCR特异性STAT3α和β剪接变体，接着的产品切割用限制酶特异于在S剪接变体， 阿飞探索两种剪接事件之间的关联。产品的密度表明包容Ser701为roughly十倍比其在两个STAT3α和β遗漏（ΔS）更常见（数据未显示）。然而，这种半定量方法是不能充分再现的，并不能有效地用于测量所有四个剪接同时变体。分析每一个的四个剪接变异体的比例，有必要建立产生紧技术（给定样品的数测定）定量PCR（qPCR的）协议复制。 相对定量PCR依赖于感兴趣的基因的比较已知在特定水平20待表达标准或看家基因和当感兴趣和管家基因的基因都具有相似的效率扩增是适当的。双链（DS）DNA结合的荧光（花青）染料结合的PCR扩增子21，并有一定数目的循环后，足够的扩增已发生荧光是可检测的。较高的初始水平谈话中，较低的阈值循环（CT）值。自cDNA制备的浓度不同，需要的转录物的浓度与已知在所有样品中一致的水平待表达的转录物，如葡萄糖醛酸酶-β（GUSB）的嗜酸性粒细胞22的浓度进行比较。 相对的qPCR不是高度相似的序列可行，如在从串联剪接产生的剪接变体。特异性扩增剪接变体所需的严格条件导致效率下降。相反，绝对定量必须使用23。这需要制备标准曲线用感兴趣的剪接转录的已知浓度，并确保PCR条件优化特异性24。如所描述的，对于特定的基因绝对和相对的qPCR数据可合并以告知该基因的表达的理解在特定的细胞类型，在这种情况下，STAT3在各种刺激嗜酸性粒细胞25。 这里，STAT3剪接变体定量与期望该方法可以适用于其它串列剪接事件的有针对性的研究说明。优化是一个漫长的过程，在不同浓度和循环参数无数次的迭代几对引物在几个月的过程中进行测试。该协议的主要特点是基于与剪接变异体已知浓度的标准曲线引物特异性验证和量化。结合相对定量PCR证明了我们的应用程序帮助，但不是必需的。

<table><tbody><tr><td>MJ Research PTC-200 Thermal Cycler</td><td>GMI</td><td>N/A</td><td>Used for standard PCR</td></tr><tr><td>7500 Real-Time PCR System</td><td>Applied Biosystems</td><td>N/A</td><td>qPCR machine</td></tr><tr><td>GS-6R</td><td>Beckman Coulter</td><td>N/A</td><td>centrifuge for 96-well plates</td></tr><tr><td>Nanodrop 2000 sprectrophotometer</td><td>ThermoFisher Scientific</td><td>N/A</td><td></td></tr><tr><td>RPMI-1640 medium</td><td>Sigma Aldrich</td><td>R8758</td><td>cell culture medium</td></tr><tr><td>PfuTurbo DNA Polymerase</td><td>Agilent Technologies</td><td>600410</td><td>DNA polymerase for standard PCR</td></tr><tr><td>KpnI</td><td>New England Biosciences</td><td>R0142S</td><td></td></tr><tr><td>NheI</td><td>New England Biosciences</td><td>R0131S</td><td></td></tr><tr><td>SYBR Green PCR Master Mix</td><td>Qiagen</td><td>330523</td><td>qPCR, DNA polymerase/dsDNA-binding dye mix</td></tr><tr><td>Rneasy Mini Kit</td><td>Qiagen</td><td>74204</td><td>RNA extraction kit</td></tr><tr><td>SuperScript III First-Strand Synthesis System</td><td>Invitrogen (ThermoFisher Scientific)</td><td>18080-044</td><td>cDNA synthesis kit</td></tr><tr><td>Primers</td><td>Integrated DNA Technology</td><td>N/A</td><td></td></tr><tr><td>NEBuffer 1.1</td><td>New England Biosciences</td><td>B7201S</td><td></td></tr><tr><td>GenePure LE Agarose</td><td>ISC BioExpress</td><td>E-3120-500</td><td>component of TAE gel</td></tr><tr><td>Pipettors</td><td>Major lab suppliers (MLS)</td><td>N/A</td><td></td></tr><tr><td>Filter pipette tips</td><td>Neptune Scientific</td><td>BT10XL, BT20, BT200</td><td></td></tr><tr><td>EU One Piece Thin Wall Plate</td><td>MidSci</td><td>ABI7501</td><td></td></tr><tr><td>ThermalSeal A Sealing Film</td><td>MidSci</td><td>TSA-100</td><td>96 well plate seal</td></tr><tr><td>pET-Elmer (variant of pET-28a)</td><td>Novagen; modified in Mosher lab</td><td>N/A</td><td>Details in PMID: 20947497</td></tr><tr><td>Wizard Plus SV Minipreps DNA purification system</td><td>Promega</td><td>A1460</td><td>Plasmid purification</td></tr><tr><td>BigDye Terminator v3.1 Cycle Sequencing Kit</td><td>ThermoFisher Scientific</td><td>4337455</td><td>Sequencing kit</td></tr><tr><td>QIAEX II Gel Extraction kit</td><td>Qiagen</td><td>20021</td><td>Amplicon purification</td></tr><tr><td>DH5&amp;alpha; competent cells</td><td>ThermoFisher Scientific</td><td>18265-017</td><td>available from several providers, see PMID: 2162051</td></tr><tr><td>kanamycin</td><td>Research Products International Corp.</td><td>K22000-5.0</td><td></td></tr><tr><td>Tris base</td><td>ThermoFisher Scientific</td><td>BP152-5</td><td>component of TAE buffer</td></tr><tr><td>Acetic acid, glacial</td><td>ThermoFisher Scientific</td><td>A38C-212</td><td>component of TAE buffer</td></tr><tr><td>EDTA (Ethylenediaminetetraacetic acid)</td><td>Sigma Chemical Company (Sigma Aldrich)</td><td>E-5134</td><td>component of TAE buffer</td></tr><tr><td>Bacto Tryptone</td><td>BD Biosciences</td><td>211705</td><td>component of Luria Broth</td></tr><tr><td>Bacto Yeast extract, technical</td><td>BD Biosciences</td><td>288620</td><td>component of Luria Broth</td></tr><tr><td>Sodium chloride</td><td>ThermoFisher Scientific</td><td>S271-10</td><td>component of Luria Broth</td></tr><tr><td>Sodium hydroxide</td><td>ThermoFisher Scientific</td><td>SS255-1</td><td>component of Luria Broth</td></tr><tr><td>Bacto Agar</td><td>BD Biosciences</td><td>214010</td><td>component of Luria Broth plate</td></tr><tr><td>Lasergene SeqBuilder</td><td>DNASTAR</td><td></td><td>Figure 1 generated using Lasergene SeqBuilder software version 12.2.0 (DNASTAR)</td></tr></tbody></table>

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.

质量好的定量PCR数据会产生一个S形扩增曲线（ 图2a），表示在循环的过程中成绩单呈指数增长。过分模板的存在可导致高荧光背景，这意味着不恰当的基线是建立在最初的几个循环。如果数据不提供的指数曲线（ 图2b），进一步优化是必要的（步骤3.1和3.4所述）。有关解决qPCR的结果进一步信息，请参考32。为模板校准质粒产生的标准曲线将指示的扩增（ 图3中所示为STAT3Sα曲线）的效率。所述的条件下，观察83至95％之间的效率。为特异性（步骤3.4）的公式假定相等的效率，这是不可能的25，这样的特异性很可能是大于特异性因子暗示。 为了评估STAT3水平的一致性，测定四个剪接变体的绝对值以及总STAT3，后者使用引物扩增的区域共同所有四个剪接变体（ 图4）的水平。理想情况下，线性回归（相关指示）和斜率（泛STAT3来概括剪接变异体的比率）都应该是接近1。 绝对的qPCR数据表示为饼图显示四个剪接变体的比例随时间刺激后用细胞因子（ 图5a）。嗜酸性粒细胞静息（0小时）有STAT3Sα的比例最小，但这种变异一直是最丰富的。相乘的STAT3β剪接变体的级分（S ^6 +ΔSβ）通过STAT3ΔS剪接变体的级分（ΔSα+ΔSβ）一致地得到比ΔSβ实验上记录的值低的值。如果剪接事件是独立的，人们会预期相乘ΔS的分数由β变体的级分变体将给与该实验确定的值一致的值。不是从独立的剪接预计ΔSβ的更高水平的建议一个共同拼接存在偏差。 合并绝对和相对定量PCR数据表明，所有STAT3剪接变异体水平升高刺激后细胞因子IL-3和TNFα，含量达到峰值6小时后刺激（ 图5b - E）。三四个剪接变异体，转录水平相比，在媒体嗜酸性粒细胞IL-3 +TNFα治疗嗜酸性粒细胞（6小时）高出约3倍在同一时间点。STAT3Sα水平在IL3 +TNFα治疗嗜酸性粒细胞的3.5倍相比，在该时间点在媒体嗜酸性粒细胞。最大的不确定性（最大的标准误差测量的）被认为在ΔSβ（ 图5e），其包括总STAT3的所有样品中的最小的部分。这并不奇怪，因为较低水平与较高的Ct值相关联。需要更多的周期，以达到所述阈值周期将化合物的不确定性是由于扩增效率周期到周期的变化。 <img alt="图1" src="/files/ftp_upload/54473/54473fig1.jpg" /> 图1: 引物对原理用于执行STAT3剪接变异体和泛STAT3用于特异性地扩增每个STAT3剪接变异体（Sα，Sβ，分别ΔSα和ΔSβ）的引物有s 的定量PCR。hown。正向引物（STAT3"S"和"ΔS"），跨越外显子21和22之间的交界处<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473fig1large.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/54473/54473fig2.jpg" /> 图 2: 的qPCR数据的扩增曲线 （ 一 ）S形扩增曲线装置可靠扩增。从定量PCR获得含有STAT3Sα质粒两个连续稀释的这些数据，每一对代表重复稀释的样品的以上的40个循环（x轴）的过程中荧光水平彩色线条。最浓的样品（绿 - 灰色）充分通过循环17放大（与双链DNA结合染料成正比的荧光，在y轴上示出），以超过阈荧光值（baseli显示甲肾上腺素绿色箭头）。其Ct值是17，（b）非指数曲线表明背景荧光阈没有被正确建立的前几个周期。这可能是由于抑制剂，或高度浓缩的模板或引物的存在。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473fig2large.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/54473/54473fig3.jpg" /> 图3: 日志的标准曲线 VS 的Ct（STAT3Sα 的拷贝数 ），还有就是每个日志STAT3剪接变异体的拷贝数和循环阈值（CT）之间的线性关系。创建从质粒DNA的标准曲线模仿样品的cDNA和由此提供比稀释PCR扩增子创建了一个曲线更好的测量。给出的数据是由定量PCR含有STAT3Sα质粒两个连续稀释液获得的Ct值。从该曲线，存在于每个样品中的拷贝数可被内插，并且扩增效率计算（83.9％）。虽然y轴截距比坡度小于再现的，截距表明42.2循环将有必要是一定的无靶DNA存在。误差棒表示SEM，每组分别构建了STAT3Sβ，ΔSα和ΔSβ（未显示）3.可比曲线。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473fig3large.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/54473/54473fig4.jpg" /> 图4: 量化泛比较 <s仲&gt; STAT3 VS累计 STAT3 剪接变异体。加入STAT3剪接的变种回归VS STAT3总应该有坡度（泛的累积比率）和R 2的值（相关性）接近1的值从17个样品（嗜酸性粒细胞和DLBCL）包括在内。误差线表示的x到-Y测定的SEM，N≥2每个。图改编自参考20。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54473/54473fig5.jpg" /> 图 5:STAT3 剪接变异体水平波动过细胞因子治疗过程 （ 一 ）饼图表示每个STAT3剪接变异体的百分比在IL3和TNFα治疗过程中嗜酸性粒细胞。 （ 二 - 五 ）的变化的STAT3剪接变体在嗜酸性粒细胞与细胞因子的各种组合，通过组合的相对和绝对的qPCR数据测量处理的 STAT3Sα（b）中 ，Sβ（c）中 ，ΔSα（d）和ΔSβ（E）的水平。波动一段时间后的刺激。最初的水平增加，调峰6小时刺激后。该IL3 +TNFα引起的组合比单独IL3所有四个STAT3剪接变异体的高表达。 SEM每个数据点占误差传播计算。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54473/54473tbl1.jpg" /> 表格1:<html>用于扩增（a）中 ，绝对的（b）和相对的（ 三 ）定量PCR的引物。克隆引物有限制序列和5&#39;-扩展高效切割。KpnI和NheI位限制性酶切位点以粗体表示。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473tbl1large.jpg" target="_blank">请点击这里查看此表的放大版本。</a> <img alt="表2" src="/files/ftp_upload/54473/54473tbl2.jpg" /> 表2. PCR循环参数（左）和试剂卷（右）扩增（一），相对的（B），绝对（三）定量PCR。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473tbl2large.jpg" target="_blank">请点击这里查看此表的放大版本。</a> <html> 3 / 54473tbl3.jpg"/&gt; 表3:模板绝对定量PCR质粒校准测定该测定是必要的，以评估再现性和效率，以及产生从中插数据的标准曲线。 "非目标"混音会给特异性的估计。优化可能需要实现的一致性。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473tbl3large.jpg" target="_blank">请点击这里查看此表的放大版本。</a> <img alt="表4" src="/files/ftp_upload/54473/54473tbl4.jpg" /> 表4:模板相对的qPCR（泛 STAT3 和管家基因 GUSB）校准测定不同于绝对定量PCR，该测定的点是确定在哪些扩增效率是〜100％的条件。tp_upload / 54473 / 54473tbl4large.jpg"目标="_空白"&gt;请点击这里查看此表的放大版本。 <img alt="表5" src="/files/ftp_upload/54473/54473tbl5.jpg" /> 表5:。模板测量泛 STAT3 和看家基因 GUSB 相对定量PCR检测样品的标准曲线的样品重复在一起，以保证检测的效率相媲美<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473tbl5large.jpg" target="_blank">，请点击这里查看此表的放大版本。</a> <img alt="表6" src="/files/ftp_upload/54473/54473tbl6.jpg" /> 表6:模板，以测量S变种绝对定量PCR检测样品的标准曲线的样品重复在一起，以保证可比性EFF。<html>该试验的iciency。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473tbl6large.jpg" target="_blank">请点击这里查看此表的放大版本。</a> <img alt="表7" src="/files/ftp_upload/54473/54473tbl7.jpg" /> 表7:。模板测量 ΔS变种 绝对定量PCR检测样品的标准曲线的样品重复在一起，以保证检测的效率相媲美<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54473/54473tbl7large.jpg" target="_blank">，请点击这里查看此表的放大版本。</a> <img alt="表8" src="/files/ftp_upload/54473/54473tbl8.jpg" /> 表8:模板测量泛 STAT3 绝对定量PCR样品检测 。large.jpg"目标="_空白"&gt;请点击这里查看此表的放大版本。

Watch this Scientific Journal Video about Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts at JoVE.com

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

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 ...

merging-absolute-relative-quantitative-pcr-data-to-quantify-stat3

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Additional Roles of RNA

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14.8K 次观看.  University of Wisconsin-Madison. 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 α/β C-termini, can be quantified.

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

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

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

遗传学

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

使用纳米流体技术的单体和散装 C. 电子样品 中的高通量定量 RT-PCR

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

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 ...

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

ACT1-CUP1测定确定出芽酵母中剪接体突变体的底物特异性敏感性

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

医学

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

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

检测选择性剪接的过程中上皮 - 间质转化

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

生物学

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 ...

3' End Sequencing Library Preparation with A-seq2

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3&#39;UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3&#39; end processing. Also this current protocol takes advantage of the polyadenylate (poly(A)) tails that are added during the biogenesis of most mammalian mRNAs to enrich for fully processed mRNAs. A DNA adaptor with deoxyuracil at its fourth position allows the precise processing of mRNA 3&#39; end fragments for sequencing. Not including the cell culture and the overnight ligations, the protocol requires about 8 h hands-on time. Along with it, an easy-to-use software package for the analysis of the derived sequencing data is provided. A-seq2 and the associated analysis software provide an efficient and reliable solution to the mapping of pre-mRNA 3&#39; ends in a wide range of conditions, from 106 or fewer cells.

This protocol describes a method for mapping pre-mRNA 3&#39; end processing sites.

合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

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

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

使用纳米流体技术的单体和散装 C. 电子样品中的高通量定量 RT-PCR

使用 E1A 微型工具研究 mRNA 拼接更改

ACT1-CUP1测定确定出芽酵母中剪接体突变体的底物特异性敏感性

脑脊液的microRNA分析采用实时定量PCR技术

一种基于报告基因的细胞检测，用于监测剪接效率

检测选择性剪接的过程中上皮 - 间质转化

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

3 A-seq2 的末端测序库的研制

合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

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

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

使用纳米流体技术的单体和散装 C. 电子样品 中的高通量定量 RT-PCR

使用 E1A 微型工具研究 mRNA 拼接更改

ACT1-CUP1测定确定出芽酵母中剪接体突变体的底物特异性敏感性

脑脊液的microRNA分析采用实时定量PCR技术

一种基于报告基因的细胞检测，用于监测剪接效率

检测选择性剪接的过程中上皮 - 间质转化

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

3 A-seq2 的末端测序库的研制

使用纳米流体技术的单体和散装 C. 电子样品中的高通量定量 RT-PCR