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&#8217;s Phosphate-Buffered Solution</td>
			<td>Corning</td>
			<td>21-030</td>
			<td>1X PBS.</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma&#160; 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&#160; 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&#160; reverse transcription of mRNA into&#160; cDNA synthesis. Kit includes&#160; 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&#160;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&#160; 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&#160;Particle&#160;Counter</td>
			<td>Beckman Coulter</td>
			<td>6605698</td>
			<td>Particle counter. For counting cells before plating&#160; 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

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

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

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

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

合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

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

科研

遗传学

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

工程人为因素来特异性地操纵人细胞中的选择性剪接

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

反向转录-环介导的等温放大 (RT 灯) 测定尿液、血清和蚊子标本中的 Zika 病毒和管家基因

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)&#8212;we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-&#954;B pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers&#8212;as it portends a significant roadblock against immunotherapy&#8212;the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

慢性CDK9抑制的基于小鼠细胞系的模型，研究癌症中普遍存在的非遗传转录伸长缺陷（TE肯定）

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription ...

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the others contain 5&#39; monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5&#39; triphosphate. However, the triphosphate at primary 5&#39; termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5&#39; polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5&#39; triphosphate.
In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5&#39; polyphosphatase, which converts 5&#39; triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5&#39; end and is insensitive to 5&#39; polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.
By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5&#39; polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5&#39; triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

使用基于循环 RT-PCR 的策略对玉米 Mitochondrion 中初级和已处理转录的区分和映射

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3&#39; end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.

Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR

RNA hairpins and loops can function as primers for reverse transcription (RT) in absence of sequence-specific primers, interfering with the study of overlapping antisense transcripts. We have developed a technique able to identify strand-specific RNA, and we have used it to study HIV-1 antisense protein ASP.

通过斯特兰德特异性RT-PCR检测患者人类免疫缺陷病毒类型1（HIV-1）反义蛋白（ASP）RNA转录本

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). ...