我们想感谢 Bettine 吉布斯的技术帮助。

在执行本协议中的所有步骤时, 始终佩戴实验室大衣、手套和安全眼镜。确保含有苯酚和异硫氰酸胍试剂的容器/管只在经认证的罩盖中打开, 并在指定容器中处理苯酚废料。使用 DNAse/RNAse 无菌管, 提示和试剂。
1. 细胞培养
<ol><li>生长的 HeLa 细胞线和两个悬浮地幔细胞淋巴瘤细胞系, Granta-519 和 Jeko-1, 在 DMEM 含有10% 血清和 100 U/毫升青霉素/链霉素在一个湿润的孵化器与 5% CO2在37°c。使用粒子计数器14稀释单元格1:10 并计数。</li>
	<li>用于 RNA 提取, 板50万细胞/井在6井板 (每井2毫升介质)。孵化24小时后, 从细胞中收集 RNA。</li>
</ol>2. RNA 提取
<ol><li>收获细胞 注: 除离心步骤外, 在组织培养罩中执行所有列出的步骤以保持不孕。<ol>
<li>对于悬浮细胞 (Jeko-1 和 Granta-519):<ol>
<li>将细胞 (连同2毫升介质) 转移到15毫升管, 离心机在 1725 x g 处进行5分钟的抽吸培养基并将细胞颗粒留在管的底部。</li>
			<li>并用重悬50µL 磷酸盐缓冲盐水 (PBS) 中的细胞颗粒。在1.5 毫升离心管中添加500µL 单相苯酚和异硫氰酸胍试剂到每个样品中。混合好, 并在室温下 (RT) 离开5分钟, 同时反转离心管每1分钟。</li>
		</ol>
</li>
		<li>对于黏附细胞 (HeLa):<ol>
<li>从每个井中吸入细胞培养介质。每井加1毫升的 PBS, 以洗涤残骸。</li>
			<li>去除 PBS, 并在每个井中加入500µL 单相苯酚和异硫氰酸胍试剂。</li>
			<li>在 RT 中孵育5分钟, 轻柔摇摆。转到1.5 毫升离心管。</li>
		</ol>
</li>
	</ol>
</li>
	<li>细胞裂解<ol>
<li>将苯酚和异硫氰酸胍细胞混合物冷冻-20 摄氏度, 1 小时。 注: 混合可在一夜之间留在-20 摄氏度或直到需要。</li>
		<li>在冰上解冻苯酚和异硫氰酸胍细胞混合物。在 PCR 罩中添加100µL 氯仿到离心管。涡流样品 10-十五年代, 直到混合物是粉红色和不透明的。在冰上孵化样品 (摄氏4摄氏度), 15 分钟。</li>
		<li>离心样品为15分钟在 20800 x g 和4°c。小心移除上无色的水相 (~ 200 µL) 和转移到一个新的1.5 毫升离心管。</li>
	</ol>
</li>
	<li>RNA 降雨雪<ol>
<li>在每个样品中加入相同数量的异丙醇。添加1µL 的 coprecipitant (参见材料表), 以作为 rna 的载体, 并帮助在后续步骤中可视化 rna。将样品孵育-80 摄氏度, 至少4小时。为了取得最佳效果, 在-80 摄氏度的夜间孵化。</li>
		<li>将样品转移到冷却离心机上。离心机为35分钟, 在 20800 x 克/4 °c;RNA 会出现在管子底部的蓝色斑点上。小心地从样品中取出异丙醇。添加500µL 80% 乙醇和并用重悬颗粒由简要涡流 3 s。</li>
		<li>离心样品在 20800 x g 在 RT 5 分钟. 从样品中取出所有的乙醇。让样品空气干燥约10分钟并用重悬样品在35µL 的 RNAse 无水。放置在热块 (65 °c) 为5分钟, 以确保 RNA 完全在溶液中, 并立即将管放在冰上。</li>
		<li>使用以前描述的分光光度计确定总 RNA 的浓度, 除非使用 RNA 样本的1.5 µL, 而不是1µL15。可以选择在1% 琼脂糖凝胶上运行1µg 的总 rna, 以确定 rna 的完整性。</li>
	</ol>
</li>
</ol>3. DNase 治疗
<ol><li>在对 rna 进行量化后, 对总 rna 进行 DNAse 处理, 以降解任何基因组 DNA。对于每个样品, 添加以下试剂从无 RNAse DNAse 试剂盒, 使20µL 总反应混合在一个单独的离心管:<ol>
<li>混合2µL 的 DNase 10X 反应缓冲器与4.4 µg 的总 RNA。带水共14µL。</li>
		<li>添加2µL 的无 RNase DNAse。在2µL RNase 抑制剂 (从反向转录试剂盒) 存在的情况下, 在37摄氏度孵育30分钟。</li>
		<li>添加2µL 的停止溶液和热量在一个热量块10分钟70°c, 以使 DNAse 酶失效。 注: DNAse 治疗是可选的。</li>
	</ol>
</li>
</ol>4. cDNA 合成
为最后反应容量50µL:
<ol><li>对于最终的反应体积为50µL: 转移22µL 的 DNase 处理的 rna 到一个新的管, 或转移4µg 的总 RNA 与增加的水到总容量22µL 到一个新的管子。从10µM 底漆溶液中添加2µL T7 寡核苷酸25底漆。T7 寡核苷酸25底漆的序列为 5 '-GCCGGTAATACGACTCACTATAGTTTTTTTTTTTTTTTTTTTTTTTTT-3 '。</li>
	<li>从反向转录套件添加2µL 的 RNase 抑制剂 (20 U/µL), 8 µL 的5x 反应缓冲器, 4 µL 10 毫米 dNTPs, 和2µL 的逆转录酶 (200 U/µL)。 注意: 建立一个没有反向逆转录酶的反应作为一个阴性控制。</li>
	<li>孵育在42°c 1 小时直接转移到冰。把管子加热75摄氏度5分钟, 然后将管子放在冰上。</li>
</ol>5. 融合基因转录的底漆搜索
<ol><li>
底漆设计
	<ol>
<li>从集成基因组浏览器 (ENST00000360839.6/NM_017747) 下载 cDNA 序列, 并在停止密码器上游的目标记录 (ANKHD1) 的 ORF 中标识一个区域。复制并粘贴整个区域 (最多700核苷酸), 其中包含停止密码子的上游序列进入底漆设计软件 (< https://www.idtdna.com/Primerquest/Home/Index >) 的序列输入区域。</li>
		<li>选择 "显示自定义设计" 选项, 然后选择 "10" 以确定系统应生成的引物数 (返回结果), 并将所有其他参数设置为默认值。</li>
		<li>选择五向前底漆和两个不重叠的反向底漆。命令引物和重建与 RNAse/DNAse 水的最终浓度为10µM。有关 PCR 的一般底漆设计的更多详细信息将在其他16中介绍。</li>
	</ol>
</li>
	<li>
PCR 识别潜在引物用于随后的 3 ' 种族 注: 为了确定在反应中使用的最终正向底漆, 通过在 pcr 罩中设置不同的正向和反向引物组合, 对所设计的底漆进行测试。<ol>
<li>将 cDNA (2 µL) 转移到新鲜的0.5 毫升 PCR 管。添加1µL 的前底漆和1µL 的反向底漆 (从10毫米底漆溶液)。通过添加8.5 µL 的核酸酶水, 使12.5 µL。添加12.5 µL 的 2x PCR 到凝胶主混合。</li>
		<li>使用以下 PCR 热循环条件:95 °c 为2分钟跟随29个周期95°c 为三十年代, 60 °c 三十年代和72°c 为 1 min. 设置最后周期在68°c 为7分钟。</li>
		<li>PCR 后, 运行的产品在1% 琼脂糖凝胶染色的溴溴。电泳的详细信息如前所述, 有一些修改17。<ol>
<li>在250毫升的烧瓶中, 将1克琼脂糖溶于100毫升的三醋酸 (泰) 缓冲器中。用微波炉煮。</li>
			<li>离开冷却1分钟, 并添加7.5 µL 的溴溴化溶液 (10 毫克/毫升股票) 在一个通风罩。将烧瓶旋转, 倒入卧式凝胶装置, 搅拌均匀。在通风罩的 RT 中留出至少30分钟, 使凝胶凝固。</li>
			<li>用1x 泰式缓冲液盖住凝胶, 将 PCR 产物的10µL 加在琼脂糖凝胶上, µL 3-5 的 DNA 分子量阶梯。运行在 175 V 10 分钟. 使用成像仪可视化产品, 如果需要进一步分离产品, 则运行凝胶以增加 5-10 分钟。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
3 ' 种族的嵌套引物选择
	<ol>
<li>分析 pcr 琼脂糖凝胶的结果, 选择具有独特、强、单一 PCR 波段的引物。使用 5 '-最底漆 (ANKHD15 '-CCTTCCCAGCGAGTTTCTAC-3 ') 连同 T7 oligodT25底漆在第一次 PCR 运行。</li>
		<li>选择位于第一个底漆下游的嵌套底漆 (ANKHD1 5 '-CGTTGGACACAGTGGAATCT-3 '), 并在第二套 PCR 反应中使用 T7 底漆 (5 '-GGCCTAATACGACTCACTATAG-3 ')。</li>
	</ol>
</li>
</ol>6. 利用两种不同的酶优化 3 ' 种族来映射 3 ' UTR
注: 用于 PCR 的 DNA 聚合酶的多样性有所增加;因此, 我们想确定的标准条件, 可以应用, 即使在使用不同的酶 3 ' 种族 PCR 反应。任何成绩单的反向引物保持不变;唯一的更改在嵌套的向前引物中, 特定于目标记录。
<ol><li>
协议 1: 3 ' 种族使用改进的脱氧核糖核酸聚合酶从Pyrococcus furiosus (Pfu)
	<ol>
<li>第一 PCR<ol>
<li>将1µL 的 cDNA 转移到 PCR 管, 并添加5µL 10x Pfu反应缓冲器。添加1µL 的第一个嵌套正向底漆, 1 µL 的 T7 oligodT25底漆, µL 1 dNTPs (从 10 mM 解决方案)。</li>
			<li>增加40µL 的水, 使49µL 总体积。添加1µL 的Pfu DNA 聚合酶并混合良好。在表 1中使用 pcr 配置文件运行 pcr。</li>
		</ol>
</li>
		<li>二次 PCR<ol>
<li>将2µL 的产品从第一 pcr 转化为新的 pcr 管, 并与5µL 10x Pfu超 II 反应缓冲器混合使用。添加1µL 的第二套 PCR 底漆, 1 µL 的 T7 底漆, 1 µL dNTPs (10 毫米溶液)。</li>
			<li>增加39µL 的水, 使µL 总反应量达到49。添加1µL 的Pfu DNA 聚合酶。使用与第一个 pcr 设置相同的 pcr 配置文件 (表 1) 运行 pcr。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
协议 2: 3 ' 种族使用嵌合体 dna 聚合酶组成的 dna 结合域融合到Pyrococcus类校对聚合酶 PCR 主混合
	<ol>
<li>第一 PCR<ol>
<li>将1µL 的 cDNA 转移到 PCR 管上。添加1µL 的第一个嵌套正向底漆和1µL 的 T7 oligodT25底漆。增加22µL 的水, 使25µL。</li>
			<li>添加25µL 的 2x PCR 大师混合和混合良好。使用表 2中描述的 PCR 循环条件。</li>
		</ol>
</li>
		<li>二次 PCR<ol>
<li>将2µL 的产品从第一 pcr 转到新的 pcr 管。添加1µL 的第二嵌套 PCR 底漆连同1µL 的 T7 反向底漆。</li>
			<li>增加22µL 的水, 使25µL。添加25µL 的 2X PCR 主组合。使用与第一个 pcr 设置相同的 pcr 配置文件 (表 2) 运行 pcr。</li>
		</ol>
</li>
	</ol>
</li>
</ol>7. 验证第二次 PCR 产品的 3 ' 种族。
<ol><li>采取 5-10 µL 的第二个 PCR 产品 (以及产品从第一轮 PCRs, 如果记录是丰富的表达) 和运行在琼脂糖凝胶。设置凝胶并运行以前描述的电泳 (步骤5.2.3.1 到 5.2.3.3)。在成像仪上可视化结果。</li>
</ol>8. 产品提纯和测序
<ol><li>根据制造商的协议, 用凝胶和 PCR 片段 DNA 清理系统从二次 pcr 中纯化凝胶 pcr 产物。</li>
	<li>执行桑格测序 (或者, 将凝胶纯化的 PCR 产品样品和适当的测序底漆发送到测序实验室)。对测序数据进行分析。</li>
	<li>下载序列分析软件 (请参阅材料表) 并将跟踪文件导入软件中。使用单个纯基 QVs (质量值) 获取基本调用信息18。下载的色谱与高品质的痕迹, 以供观赏。 可选: 可使用适当的克隆试剂盒克隆纯化的产品, 并为桑格测序发送独立克隆。</li>
</ol>

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K., Govindarajan, R., Naik, S., Kumar, 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+effect+of+hairpin+structure+on+PCR+amplification+efficiency.">The effect of hairpin structure on PCR amplification efficiency.</a> Mol Biol Today. 1, 67-69 (2000).</li><li>Kalle, E., Kubista, M., Rensing, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-template+polymerase+chain+reaction.">Multi-template polymerase chain reaction.</a> Biomol Detect Quant. 2, 11-29 (2014).</li><li>Stransky, N., Cerami, E., Schalm, S., Kim, J. L., Lengauer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+landscape+of+kinase+fusions+in+cancer.">The landscape of kinase fusions in cancer.</a> Nat Commun. 5, 4846 (2014).</li></ol>

作者没有什么可透露的。

尽管大规模并行测序技术的出现, 在基因的基础上, 3 ' 种族仍然是最简单和最经济的方法, 以确定的 PAS 和核苷酸相邻的聚 (a) 尾。这里描述的适应扩展使用 3 ' 种族放大和地图序列包括部分的 ORF, 停止密码子, 和整个 3 ' UTR 的ANKHD1 mRNA 转录。3 ' 种族的一个主要优势是, 有几个小的适应, 从 3 ' 种族的产品可以克隆成其他载体, 以促进下游审讯 3 ' UTR 功能, 包括 miRNA 靶点, 稳定性检测以及其他机?...

3 ' 末端的形成是 mrna 成熟的一个关键步骤, 它包括 PAS 的前 mRNA 下游的分裂, 然后加上 250 untemplated adenines, 组成了聚 (a) 尾1,2。聚 (a) 结合蛋白 (PABP) 绑定到聚 (a) 尾, 这保护 mRNA 转录不退化, 并促进翻译1。
目前的估计表明, 70% 的人类基因有多个通过, 从而接受替代的 polyadenylation, 导致 3 ' 末端3。因此, 重要的是要确定多 (A) 尾附加到 3 ' UTR 的其余部分, 并确定任何给定的成绩单使用的考绩制度。下一代测序的出现导致了同时识别 3 ' UTRs 和成千上万基因的通过。这种测序能力的提高需要开发生物信息学算法来分析涉及 3 ' 端的替代 polyadenylation 的数据。对于 "从头" 检测或验证 PAS, 从而从大型序列数据中映射单个基因的 3 "结束", 3 ' 种族仍然是选择4,5的方法。在 3 ' 种族的 cDNA 产品中包含的序列通常只包括 3 ' UTR 的一部分, 其中包含了聚 (a) 尾、解裂部位、pas 和 pas 上游的序列。不像 PCR, 这需要设计和使用的基因特异正向和反向引物, 3 ' 种族只需要两个基因特定的嵌套前引物。因此, PCR 要求更详细地了解被放大的基因的大区域的核苷酸序列4,6。由于 3 ' 种族使用相同的反向底漆为所有 polyadenylated RNA 记录的聚 (A) 尾, 只有向前引物需要基因特异性, 因此, 只需要知识的 mRNA 的一个显着较小的区域。这使序列未完全特征化的区域放大4、7。这使得 3 ' 种族不仅用于确定一个基因的 3 ' 末端, 而且还确定和描述在 PAS 上游的大区域, 形成了 3 ' UTR 的重要部分。通过将 5 ' 种族与修改后的 3 ' 种族相结合, 其中包括 3 ' UTR 和侧翼区域的较大部分, 可以完全序列化或克隆从 5 ' 端到其 3 ' 端8的整个 mRNA 记录。
这种应用的修改 3 ' 种族的例子是最近鉴定的新的CCND1-MRCK融合基因转录从地幔细胞淋巴瘤细胞系和癌症患者。3 ' UTR 包括来自CCND1和MRCK基因的序列, 并被顽固地 miRNA 调节9。两个嵌套CCND1特定正向引物与CCND1停止密码子的相邻和下游区域互补。虽然整个转录测序连同特定的生物信息学工具可以用于检测 3 ' UTR 10 中的基因融合, 但许多实验室可能缺乏利用这项技术的财力资源或生物信息学专业知识.因此, 3 ' 种族是一个替代的诺新识别和验证的新型融合基因涉及 3 ' UTR。考虑到已报道的融合基因的数量急剧增加, 以及通过成绩单阅读, 3 ' 种族已经成为一个强大的工具, 以表征基因序列11,12。此外, 最近的研究表明, 在 3 ' UTR 和 3 ' UTR 的长度不同的序列可能影响 mRNA 转录稳定性, 本地化, 可译性和功能13。部分原因是对绘制转录的兴趣增加, 在实验室中开发的不同 DNA 聚合酶的数量有所增加。重要的是要确定哪些类型的修改可以对 3 ' 种族协议, 以利用现有的 DNA 聚合酶的曲目。
此工作报告通过使用ANKHD1中ANKHD1部分中的嵌套底漆和两个不同的 DNA 聚合酶, 调整 3 ' 种族, 以映射整个 3 ' UTR、PAS 和 3 ' 末端解切站点成绩单。

<table><tbody><tr><td>HeLa cells</td><td>ATCC</td><td>CCL-2</td><td>Cervical cancer cell line.</td></tr><tr><td>Jeko-1 cells</td><td>ATCC</td><td>CRL-3006</td><td>Mantle cell lymphoma cell line.</td></tr><tr><td>Granta-519 cells</td><td>DSMZ</td><td>ACC-342</td><td>Mantle cell lymphoma cell line.</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma Aldrich</td><td>F6178</td><td>Fetal bovine serum for cell culture.</td></tr><tr><td>Penicillin/Streptomycin</td><td>ThermoFisherScientific</td><td>15140122</td><td>Antibiotic and antimycotic.</td></tr><tr><td>GlycoBlue</td><td>Ambion</td><td>AM9545</td><td>Coprecipitant.</td></tr><tr><td>DMEM</td><td>ThermoFisherScientific</td><td>10569044</td><td>Gibco brand cell culture media with GlutaMAX.</td></tr><tr><td>Nuclease Free-Water</td><td>ThermoFisherScientific</td><td>AM9938</td><td>Ambion DNase and RNAse free water(non DEPC treated).</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Solution</td><td>Corning</td><td>21-030</td><td>1X PBS.</td></tr><tr><td>Chloroform</td><td>Sigma&amp;nbsp; Aldrich</td><td>C7559-5VL</td><td></td></tr><tr><td>2-propanol</td><td>Sigma Aldrich</td><td>I9516</td><td></td></tr><tr><td>Reagent Alcohol</td><td>Sigma Aldrich</td><td>793175</td><td>Ethanol</td></tr><tr><td>Ethidium Bromide solution</td><td>Sigma Aldrich</td><td>E1510</td><td></td></tr><tr><td>TRIzol Reagent</td><td>ThermoFisherScientific</td><td>15596026</td><td>Monophasic phenol and guanidine isothiocyanate reagent.</td></tr><tr><td>2X Extender PCR-to-Gel Master Mix</td><td>Amresco</td><td>N867</td><td>2X PCR-to-Gel Master Mix&amp;nbsp; containing loading dye used in routine quick PCR assays and primer search.</td></tr><tr><td>10mM dNTP</td><td>Amresco</td><td>N557</td><td></td></tr><tr><td>RQ1 RNase-Free DNase</td><td>Promega</td><td>M6101</td><td>Dnase treatment kit.</td></tr><tr><td>Gel Loading Dye Orange (6X)</td><td>New England BioLabs</td><td>B7022S</td><td></td></tr><tr><td>2X Phusion High-Fidelity PCR Master Mix with HF buffer</td><td>ThermoFisherScientific</td><td>F531S</td><td>2X PCR MasterMix containing a chimeric DNA polymerase consisting of a DNA binding domain fused to a Pyrococcus-like proofreading polymerase and other reagents.</td></tr><tr><td>PfuUltra II Fusion HS DNA polymerase</td><td>Agilent Technologies</td><td>600670</td><td>Modified DNA Polymerase from Pyrococcus furiosus (Pfu).</td></tr><tr><td>RevertAid RT Reverse Transcription Kit</td><td>Thermo Fischer</td><td>K1691</td><td>Used for&amp;nbsp; reverse transcription of mRNA into&amp;nbsp; cDNA synthesis. Kit includes&amp;nbsp; Ribolock RNAse inhibitor, RevertAid M-MuLV reverse transcriptase and other reagents listed in manuscript.</td></tr><tr><td>Pefect size 1Kb ladder</td><td>5 Prime</td><td>2500360</td><td>Molecular weight DNA ladder.</td></tr><tr><td>Alpha Innotech FluorChem Q MultiImage III</td><td>Alpha Innotech</td><td></td><td>Used to visualise ethidium bromide stained agarose gel.</td></tr><tr><td>Low Molecular Weight Ladder</td><td>New England BioLabs</td><td>N3233L</td><td>Molecular weight DNA ladder.</td></tr><tr><td>Vortex Mixer</td><td>MidSci</td><td>VM-3200</td><td></td></tr><tr><td>Mini Centrifuge</td><td>MidSci</td><td>C1008-R</td><td></td></tr><tr><td>Dry Bath</td><td>MidSci</td><td>DB-D1</td><td></td></tr><tr><td>NanoDrop 2000C</td><td>ThermoFisherScientific</td><td>ND-2000C</td><td>Spectrophotometer.</td></tr><tr><td>Wide Mini-Sub&amp;nbsp;Cell GT Horizontal Electrophoresis System</td><td>BioRad</td><td>1704469</td><td>Electrophoresis equipment-apparatus to set up gel</td></tr><tr><td>PowerPac Basic Power Supply</td><td>BioRad</td><td>1645050</td><td>Power supply for gel electrophoresis.</td></tr><tr><td>Agarose</td><td>Dot Scientific</td><td>AGLE-500</td><td></td></tr><tr><td>Mastercycler Gradient</td><td>Eppendorf</td><td>950000015</td><td>PCR thermocycler.</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5430R</td><td></td></tr><tr><td>Wizard SV Gel and PCR Fragment DNA Clean-Up System</td><td>Promega</td><td>A9281</td><td></td></tr><tr><td>Zero Blunt TOPO PCR Cloning Kit, with One Shot&amp;nbsp; TOP10 Chemically Competent E. coli cells</td><td>ThermoFisherScientific</td><td>K280020</td><td></td></tr><tr><td>MyPCR Preparation Station Mystaire</td><td>MidSci</td><td>MY-PCR24</td><td>Hood dedicated to PCR work.</td></tr><tr><td>Hamilton SafeAire II fume hood</td><td>ThermoFisherScientific</td><td></td><td>Fume hood.</td></tr><tr><td>Beckman Coulter Z1&amp;nbsp;Particle&amp;nbsp;Counter</td><td>Beckman Coulter</td><td>6605698</td><td>Particle counter. For counting cells before plating&amp;nbsp; for RNA extraction.</td></tr><tr><td>Applied Biosystems Sequence Scanner Software v2.0</td><td>Applied Biosystems (through ThermoFisherScientific)</td><td></td><td>Software to analyze Sanger sequencing data.</td></tr></tbody></table>

adapting 3' rapid amplification of cdna ends to map transcripts in cancer

真核基因的成熟涉及 3 ' 端形成, 这涉及到增加一个聚 (a) 尾。为了图 3 ' 末端的基因, 传统的选择方法是 3 ' 快速扩增的 cDNA 末端 (3 ' 种族)。3 ' 种族协议需要仔细设计和选择嵌套底漆在 3 ' 未翻译的区域 (3 ' UTR) 的目标基因感兴趣。然而, 通过一些修改, 该协议可用于包括整个 3 ' UTR 和序列在开放阅读框架 (ORF), 提供了一个更全面的图片与 3 ' UTR 之间的关系。这是除了识别的 polyadenylation 信号 (PAS), 以及分裂和 polyadenylation 网站提供的常规 3 ' 种族。扩展 3 ' 种族能发现异常的 3 ' UTRs, 包括基因融合在 3 ' UTR 之内, 并且序列信息可以被使用预测潜在的 miRNA 结合的站点和不稳定的元素可能影响成绩单的稳定。

嵌套正向底漆搜索:
琼脂糖凝胶来自图 1显示两种不同的 PCR 凝胶产品 (车道1和 2), 使用相同的正向底漆, 但不同的反向底漆。3车道有一个独特的 PCR 产品, 并有一个明显的向前和反向底漆。用于 pcr 反应的理想引物是那些给出一个独特的 pcr 产品 (车道 3)。在1和2车道上使用的正向底漆给出了最强?...

Watch this Scientific Journal Video about Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer at JoVE.com

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

3 ' 快速扩增 CDNA 末端, 以图记录癌症

在这里描述的两种不同的 3 ' 快速扩增的 cDNA 结束 (3 ' 种族) 协议使用两种不同的 DNA 聚合酶来映射序列, 包括开放阅读框架 (ORF) 的一部分, 停止密码子, 和整个 3 ' UTR 的成绩单使用 RNA从不同的癌细胞系获得的。

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

adapting-3-rapid-amplification-cdna-ends-to-map-transcripts

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

12.2K 次观看.  Butler University. 在这里描述的两种不同的 3 ' 快速扩增的 cDNA 结束 (3 ' 种族) 协议使用两种不同的 DNA 聚合酶来映射序列, 包括开放阅读框架 (ORF) 的一部分, 停止密码子, 和整个 3 ' UTR 的成绩单使用 RNA从不同的癌细胞系获得的。

视频: Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

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. ...

科研

癌症研究

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

单细胞的转录组学分析

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

神经科学

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

使用转录组学分析绘制无序致癌转录因子的结构-功能关系

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.

ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells.

用于癌症表观遗传学研究的ATAC-Seq优化

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

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

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

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

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

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序

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

医学

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

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

有针对性的RNA测序分析表征基因表达和基因组改变

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

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.

3 A-seq2 的末端测序库的研制

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; ...

Rapid Amplification of cDNA Ends

Source: Pablo Sanchez Bosch2, Sean Corcoran2 and Katja Br&#252;ckner1,2,3 
1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research 
2Department of Cell and Tissue Biology,&#160; 
3Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA&#160; &#160;&#160;
Rapid Amplification of cDNA Ends (RACE) is a technique that allows amplification of full-length cDNA from mRNA by extending to the 3&#8217; or 5&#8217; end, even without prior knowledge of the sequence (Frohman et al., 1988). In contrast to regular PCR, it uses only one specific PCR primer and a second non-specific primer that will indiscriminately bind to most mRNAs. Depending on whether the area to be amplified is on the 3&#8217; or 5&#8217; end of the mRNA, the second primer is chosen to bind the polyA tail (3&#8217; end) or a synthetic linker added to all transcripts (5&#8217; end). These primer combinations are known to yield &#8220;one-sided&#8221; or &#8220;anchored&#8221; PCR due to PCR amplification using the known sequence of one side- 3&#8217; or 5&#8217;(Ohara et al., 1989). The approach allows the capture of gene-specific rare mRNAs that otherwise would be hard to detect, e.g. because of their relatively low expression or unknown complete sequence (Frohman et al., 1988).
RACE is initiated with a reverse transcription step (RT) to synthetize single stranded complimentary DNA (cDNA) from mRNA. This is followed by two consecutive PCRs that amplify the cDNA fragments from a gene of interest. To perform RACE, the sequence of the gene of interest must be at least partially known, as it is needed to design the gene-specific primers (GSPs; Frohman, 1994; Liu and Gorovsky, 1993). As the second primer pair is a generic primer that will anneal to all transcripts present in the sample, the specificity of the PCR reactions is reduced. RACE is therefore ideally performed with two nested PCRs to lower the chances of amplifying non-specific transcripts (Figure 1). Depending on the direction of amplification relative to the GSP, the technique is categorized as 5&#8217; or 3&#8217; RACE (Figure 1).&#160;
3&#8217; RACE (Figure 1A) takes advantage of the poly(A) tail present in mRNAs as a generic binding site for the non-specific primer. This simplifies the technique, as one can synthetize cDNA by using oligo (dT) primers. GSPs are located in the 5&#8217; region of the transcripts. This RACE variant allows the detection of transcript variants and different 3&#8217; UTRs (Scotto Lavino et al., 2007a). In 5&#8217; RACE (Figure 1B), GSPs are located in the 3&#8217; region of the gene. To be able to use a non-specific primer that binds to all transcripts, an adapter that serves as generic primer binding site is attached to the 5&#8217; RNA ends. To attach the adapter to the mRNA, the 5&#8217; cap that protects mRNAs against exonucleases and promotes translation must be removed (Bird et al., 2016). Opposed to 3&#8217; RACE, 5&#8217; RACE helps finding differential 5&#8217; splice variants and alternative 5&#8217; UTRs (Scotto Lavino et al., 2007b).
Here we will perform 3&#8217; RACE to identify and isolate different transcript variants using the known 5&#8217; sequence (Figure 2A) encoded by the Drosophila dSmad2 (Smox) gene. dSmad2, a fly Smad protein, is an important transducer of Activin-&#946; signaling, a pathway of the TGF-&#946; superfamily. dSmad2 regulates multiple cellular processes, such as cell proliferation, apoptosis and differentiation (Upadhyay et al., 2017). As differentially spliced transcripts of dSmad2 may have different functions in the adult fly, a first step in exploring these potential functions is to assess all transcript variants of dSmad2 at this developmental stage.

Source: Pablo Sanchez Bosch2, Sean Corcoran2 and Katja Br&#252;ckner1,2,3
1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research
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