这项工作部分是由日本机构发展癌症治疗学创新研究 (p 直接) (15cm0106102h0002、K.M.) 和癌症研究和治疗进化项目 (16cm01061150001、K.M.) 的项目所支持的。为医学研究开发, 阿曼德;武田科学基金会 (Y.M.);授予高松公主癌症研究基金 (13-24520, Y.M.);和 jsp KAKENHI 授予编号 JP16K07133 (Y.M.)。

1. 试剂设置
<ol><li>
用于细胞同步的试剂
	<ol>
<li>为了生成 Dulbecco 的改良鹰的培养基 (DMEM), 含有2.5 毫米胸苷, 溶解30.28 毫克的胸苷每1毫升的组织培养级水, 以准备125毫米胸苷。用0.22 微米注射器过滤器滤除溶液。添加1毫升新制备的125毫米胸苷每50毫升的 DMEM 补充与 10% (卷/卷) 胎牛血清 (血清)。</li>
		<li>要生成含有0.1 微克/毫升 nocodazole 的 DMEM, 请在二甲基亚砜 (亚砜) 中溶解 nocodazole, 以制备5微克/ul nocodazole。添加1µL 5 µg/µL nocodazole 每50毫升 DMEM 补充 10% (卷/卷) 的血清。</li>
		<li>要生成包含 0.002% (卷/卷) 的亚砜的 DMEM, 请添加1µL 每50毫升的亚砜 DMEM 补充 10% (卷/卷) 的血清。</li>
	</ol>
</li>
	<li>
用于 IP RdRP 测定的试剂
	<ol>
<li>制备裂解缓冲器 A: 150 毫米氯化钠, 20 毫米三盐酸 (pH 7.4) 和 0.5% (卷/卷) Nonidet P-40 (NP-40)。将试剂贮存在摄氏4摄氏度。</li>
		<li>准备5x 醋酸缓冲液:50 毫米 2-[4-(2-羟乙基)-1-哌嗪基] 磺酸 (HEPES)-KOH (pH 7.8), 500 毫米醋酸钾和20毫米氯化镁2。在室温下贮存试剂。</li>
		<li>准备洗涤缓冲器 1: 1x 醋酸缓冲液, 10% (卷/卷) 甘油, 0.1% (卷/卷) 海卫 X-100 和0.06x 蛋白酶抑制剂鸡尾酒, ethylenediamenetetraacetic 酸 (EDTA)-免费。在使用日或使用前一天准备洗涤缓冲器1。将试剂贮存在摄氏4摄氏度。</li>
		<li>准备 AGC 解决方案: 1x 醋酸缓冲液, 10% (卷/卷) 甘油和 0.02% (重量/卷) 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonat (章)。将试剂贮存在摄氏4摄氏度。</li>
		<li>要生成包含 2 mM CaCl2的 agc 解决方案, 请添加100µL 1 M CaCl2每50毫升的 agc 解决方案。将试剂贮存在摄氏4摄氏度。</li>
		<li>要生成包含3毫米乙二醇双 (b-aminoethylether) n, n, n ', n '-四乙酸四酸 (EGTA) 的 agc 解决方案, 增加375µL 0.4 米 EGTA (pH 7.5) 每50毫升的 AGC 解决方案。将试剂贮存在摄氏4摄氏度。</li>
		<li>准备洗涤缓冲器 2: 1x 醋酸盐缓冲器和 0.02% (重量/卷)。将试剂贮存在摄氏4摄氏度。</li>
		<li>准备显示器解决方案: 0.2 米 HEPES (pH 7.8), 40 毫米氯化镁2和2毫米 dithiothreitol (德勤)。将试剂贮存在摄氏-20 摄氏度。 注: 头盔的解决方案至少应在三月内更新。</li>
		<li>制备2x 蛋白酶 K 缓冲器:20 毫米三盐酸 (pH 7.6), 20 毫米 EDTA 和 1% (重量/卷) 月桂酸钠 (SDS)。将试剂贮存在摄氏-20 摄氏度。</li>
		<li>准备2x 加载缓冲器: 95% (卷/卷) 甲酰胺, 20 毫米 EDTA 和 1% (重量/卷) 橙 g. 将试剂贮存在-20 摄氏度。</li>
	</ol>
</li>
</ol>2. HeLa 细胞的制备
注: 制备 HeLa 细胞在有丝分裂阶段 (操作) 同步或异步分裂 (unmanipulated)。文化细胞在存在 5% CO2在37°c。
<ol><li>
HeLa 细胞在有丝分裂中的同步
	<ol>
<li>板 1 x 106的 HeLa 细胞与10毫升的 DMEM 补充 10% (卷/卷) 血清在10厘米培养皿。</li>
		<li>电镀后两天, 用10毫升的 DMEM 取代培养基, 其中含有2.5 毫米胸苷和 10% (卷/卷) 的血清。培养细胞24小时。</li>
		<li>用10毫升磷酸盐缓冲盐水 (PBS) (-) 冲洗细胞三次, 并培养出10毫升 DMEM 的细胞, 含 10% (卷/卷) 的血清为 6 h。</li>
		<li>用10毫升的 DMEM 取代培养基, 含有0.1 µg/毫升的 nocodazole 和 10% (卷/卷) 的血清, 并培养细胞为 14 h。</li>
		<li>轻轻摇动培养基, 取出含有分离有丝分裂细胞的上清液。计算 IP RdRP 检测的细胞数。</li>
		<li>离心样品在 490 x g 5 分钟在4°c, 并且放弃上清。 注: 细胞颗粒可储存在-80 摄氏度。</li>
	</ol>
</li>
	<li>
unmanipulated HeLa 细胞的制备
	<ol>
<li>板 1 x 106的 HeLa 细胞与10毫升的 DMEM 补充 10% (卷/卷) 血清在10厘米培养皿。</li>
		<li>电镀后两天, 用含有 10% (卷/卷) 血清的10毫升 DMEM 取代培养基。培养细胞30小时。</li>
		<li>用10毫升的 DMEM 取代培养基, 其中含有 0.002% (卷/卷) 亚砜和 10% (卷/卷) 的血清, 并将细胞培养为 14 h。</li>
		<li>用含有1毫米 EDTA 的0.7 毫升胰蛋白酶 (2.5 克/升) trypsinization 收集细胞。计算 IP RdRP 检测的细胞数。</li>
		<li>离心样品在 490 x g 5 分钟在4°c, 并且放弃上清。 注: 细胞颗粒可储存在-80 摄氏度。</li>
	</ol>
</li>
</ol>3. IP RdRP 检测
<ol><li>蛋白质 A-琼脂糖珠制备
	<ol>
<li>将亲和树脂的1毫升 (床容积500µL) 转移到1.5 毫升离心管上。离心机在 800 x g 5 分钟4摄氏度, 并丢弃上清。</li>
		<li>添加800µL 的溶解缓冲 A 到珠, 并混合通过反转管几次。离心机在 800 x g 3 分钟4摄氏度, 并丢弃上清。重复此步骤两次 (总共三洗涤)。</li>
		<li>添加800µL 的溶解缓冲 A 到珠, 混合好通过反转管几次。在旋转振动筛上一夜之间 (或至少4小时) 在4摄氏度旋转管子。</li>
		<li>离心管在 800 x g 3 分钟4摄氏度, 并丢弃上清。</li>
		<li>添加500µL 的溶解缓冲 A 到珠, 并混合通过反转管几次。把珠子存放在4摄氏度。</li>
	</ol></li>
	<li>细胞裂解剂的制备
	<ol>
<li>选如果在步骤2中冻结细胞, 将冰冻细胞放在冰上, 直到细胞松动。</li>
		<li>用10毫升的 PBS (-) 清洗细胞。离心样品在 1450 x g 5 分钟在4°c, 并且放弃上清。</li>
		<li>溶解的细胞与冰冷溶解缓冲 a (1 毫升溶解缓冲 a 每 1 x 107的细胞) 由温和的吹打。</li>
		<li>油脂实验1.5 毫升管中的样品, 条件如下;设置放大的 sonicator 在 25%, 油脂实验十年代。</li>
		<li>离心样品在 20400 x g 15 分钟在4°c。</li>
		<li>收集上清。将1毫升的上清液转化为1.5 毫升的离心管。 注意: 在无 RNase 条件下执行所有过程。</li>
	</ol></li>
	<li>免疫沉淀
	<ol>
<li>通过添加40µL (珠体积20µL), 每1毫升的裂解液中的水洗蛋白 A-琼脂糖珠, 预先清除从步骤3.2.6 的裂解液。混合好, 通过反转管几次, 并旋转样品30分钟, 在4摄氏度。</li>
		<li>离心样品在 1.3万 x g 1 分钟在4°c, 并且恢复上清。</li>
		<li>添加40µL (床容积20µL) 的水洗蛋白 A-琼脂糖珠和10µg 的抗人叔抗体 (克隆: 10E9-2)8进入预清除裂解。混合通过反转管几次, 并旋转样品在4°c 在夜间。</li>
		<li>离心样品在 3300 x g 0.5 分钟在4°c, 并且去除上清。</li>
		<li>用洗涤缓冲液清洗珠子 1: 将1毫升的洗涤缓冲器1添加到珠子上, 通过反转管混合好, 并将样品旋转5分钟, 在4摄氏度。离心样品在 3300 x g 0.5 分钟在4°c, 并且去除上清。重复此步骤三次。</li>
		<li>一次用包含2毫米 CaCl2的 agc 解决方案清洗珠, 将1毫升的 agc 解决方案加在 CaCl2到珠子上, 通过反转管混合, 并将样品旋转5摄氏度。离心样品在 3300 x g 0.5 分钟在4°c, 并且去除上清。</li>
		<li>用 Micrococcal 核酸酶 (MNase) 处理珠子: 准备表 1中描述的 MNase 反应混合物。并用重悬在60µL 的 MNase 反应混合物中的珠子由温和的吹打。轻轻摇动样品 (20 到 25 rpm) 在一个转盘上的一个在一个珀尔式孵化器在25°c 15 分钟。 注意: 使用起伏的振动筛是非常重要的, 严格按照车速限制在这一步。其他类型的振荡器, 如旋转搅拌机, 不推荐使用。</li>
		<li>离心样品在 3300 x g 0.5 分钟在4°c, 并且去除上清。</li>
		<li>用包含3毫米 EGTA 的 agc 解决方案将珠子洗两次: 添加1毫升的 agc 解决方案, 其中包含3毫米 EGTA 到珠子上, 通过反转管混合好, 并将样品旋转5分钟, 在4摄氏度。离心样品在 3300 x g 0.5 分钟在4°c, 并且去除上清。重复此步骤一次。</li>
		<li>洗涤的珠子一次与洗涤缓冲 2: 添加1毫升的洗涤缓冲 2到珠, 混合好通过反转管, 并旋转样品5分钟在4°c。离心样品在 3300 x g 0.5 分钟在4°c, 并且去除上清。在设置 RdRP 反应时, 把珠子放在冰上。 注意: 在无 RNase 条件下执行所有过程。</li>
	</ol></li>
	<li>RdRP 反应
	<ol>
<li>如表 2所述, 在新管中制备 RdRP 反应混合物。</li>
		<li>添加6µL [α-32P] UTP (3000 Ci/毫摩尔) 的混合物, 并混合良好的吹打。 注: [α-32p] ATP, [α-32p] CTP, 或 [α-32p] GTP 可用于反应, 而不是 [α-32P] UTP。</li>
		<li>将1µL 模板 RNA (1 µg/µL) 添加到混合物中, 并由吹打混合均匀。 注: 要建立 RdRP 化验, 推荐以下 RNA 模板: 5 '-GGGAUCAUGUGGGUCCUAUUACAUUUUAAACCCA-3 ' 9。</li>
		<li>并用重悬珠与20µL 的 RdRP 反应混合物从步骤3.4.3 吹打。</li>
		<li>轻轻摇动样品 (20 到 25 rpm) 在一个转台的一个转盘上的一个珀尔式孵化器为2小时在32摄氏度。</li>
		<li>在反应混合物中加入5.4 µL 蛋白酶 k (20 毫克/毫升) 和45.4 µL 2x 蛋白酶 k 缓冲液。混合通过吹打和震动 (20 到 25 rpm) 的样品在一个转盘上设置在一个珀尔式孵化器为30分钟在37摄氏度。</li>
		<li>在样品中添加109.2 µL 的 RNase 水。</li>
		<li>在样品中加入200µL 酸苯酚-氯仿。用涡旋混合, 然后在室温下将试样离心 21100 x克5 分钟。将水相转移到新管上。重复此步骤一次。</li>
		<li>添加20µL 3 米醋酸钠 (pH = 5.2), 4 µL 的沉淀载体和250µL 乙醇的水相。摇管井搅拌, 然后离心样品在 21100 x g 20 分钟在4摄氏度。放弃上清。</li>
		<li>用300µL 的冷 70% (卷/卷) 乙醇清洗颗粒, 储存在-20 摄氏度。离心样品在 21100 x g 15 分钟在4°c。</li>
		<li>丢弃上清液, 风干颗粒。 注意: 在无 RNase 条件下执行所有过程。 注: 从步骤3.4.11 的颗粒可以储存在-20 °c 至少2天。</li>
	</ol></li>
	<li>RNase 治疗
	<ol>
<li>并用重悬在20µL RNase 水中, 从步骤3.4.11 中释放出颗粒。</li>
		<li>如表 3所述, 在新管中制备 RNase 反应混合物。</li>
		<li>将 RNase 反应混合物的180µL 添加到样品中, 并在37摄氏度上孵化管2小时。</li>
		<li>添加2.3 µL 10% (卷/卷) SDS 的样本和孵化15分钟在37摄氏度。</li>
		<li>添加2µL 蛋白酶 K (20 毫克/毫升) 的样品和孵化15分钟在37摄氏度。</li>
		<li>在样品中加入205µL 酸苯酚-氯仿。用涡旋混合, 然后在室温下将试样离心 21100 x克5 分钟。将水相转移到新管上。</li>
		<li>添加20µL 3 米醋酸钠 (pH 5.2), 4 µL 的沉淀载体和250µL 乙醇的水相。摇管井搅拌, 然后离心样品在 21100 x g 20 分钟在4摄氏度。放弃上清。</li>
		<li>用300µL 的冷 70% (卷/卷) 乙醇清洗颗粒, 储存在-20 摄氏度。离心样品在 21100 x g 15 分钟在4°c。</li>
		<li>丢弃上清液, 风干颗粒。 注: 从步骤3.5.9 的颗粒可以储存在-20 °c 至少2天。</li>
	</ol></li>
	<li>凝胶电泳与显影
	<ol>
<li>并用重悬样品与20µL 的 RNase 无水。</li>
		<li>添加20µL 2x 加载缓冲区。</li>
		<li>将样品煮沸10分钟, 在95摄氏度。把样品压在冰上。</li>
		<li>运行凝胶。如果模板大小为 34 (参见步骤 3.4.3), 7 M Urea-10%polyacrylamide 凝胶用于变性凝胶电泳。</li>
		<li>用凝胶干燥器烘干凝胶。</li>
		<li>将胶片暴露在 X 光盒内的干燥凝胶中, 屏幕上的强化屏为-80 摄氏度。</li>
	</ol></li>
</ol>

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作者没有什么可透露的。

RdRP 检测是一种检测人叔 RdRP 活性的敏感方法。叔蛋白在有丝分裂 HeLa 细胞中高度表达, 其中叔形成 RdRP 复合物8,9,10。这表明有丝分裂 HeLa 细胞是检测 RdRP 活性的最佳材料。在上文所述的协议中, 有丝分裂和非同步 HeLa 细胞分别被列为阳性和阴性的例子。如图 1A所示, RdRP 从有丝分裂 HeLa 细胞中推荐的 RNA 模板中检测出的产品在20到 30 nt 之间显示出广泛的放射性信号。除了 hela 细胞外, 我们还进行了不同类型细胞系的检测, 发现信号模式可以在不同的细胞类型中改变9: 有些显示一个强信号只有大约 30 nt, 有些显示一个相似的模式与 HeLa 细胞。我们也进行了与 RNA 模板的检测, 除了 34 nt 模板8, 短和长 (300 nt) rna 与各种序列, 并成功地获得了 RdRP 产品从这些模板, 虽然可能有一些偏好。然而, 对于第一次试验, 我们建议使用 HeLa 细胞有丝分裂阶段和 34 nt RNA 模板。RdRPs 可以产生双链 RNA 两个在底漆依赖和在底漆独立方式11。我们报告, 叔保留此财产作为人类 RdRP7,9;叔合成 dsRNA 从一个 RNA 模板与 3 '-保护结构通过一个后启动机制7。对于 34 nt RNA 模板, 叔合成互补链, 不使用底漆9。为了专门检测以底漆为独立的方式合成的 RdRP 产品,即合成的 RNA 产品, [α-32p] ntp 可替换为 [γ-32p] ntp 在 RdRP 反应9。
MNase 治疗微珠上的叔免疫复合物是达到预期效果的关键步骤。如果 MNase 治疗时间过长或进行了激烈的震动, RdRP 产品将显著减少。为了避免这样的麻烦, 我们强烈建议严格遵守协议。头盔的解决方案是成功的另一个关键因素。如果您发现信号非常微弱, 请将显示器的解决方案替换为新准备的。
在整个议定书中, 人们应该小心避免 RNase 污染。核糖核酸应使用专用设备进行处理。在操纵核糖核酸后, 你应该立即丢弃提示和 RNase, 消除 RNase 与专门的解决方案 (如RNase 安静), 并改变树林。
叔与TERC和多种内源 rna 相互作用, 我们报告了其中的一部分7。在 ip-RdRP 检测中进行的修改, 如无 MNase 治疗的 ip RdRP 化验, 将提供一份完整的关于叔相关 RdRP 活动的内源 RNA 模板和生物信息学指南的序列特征。我们已经成功地将此协议应用于组织裂解。由于叔的表达在各种正常和肿瘤组织, 这可能是有益的研究非规范化的酶功能的人的叔。

人端粒酶逆转录酶 (叔) 被称为端粒细胞催化亚单位, 它拉长端粒, 使用端粒酶 rna 组分 (TERC), 特异 RNA 模板1。虽然叔聚合端粒长度 DNA 作为端粒酶的组成部分, 结构和系统发育分析表明, 叔与病毒 rna 相关的 rna 聚合酶 (RdRPs) 和病毒反转录相关系密切, 并分享域与这些聚合酶2,3,4。RdRP 是生成互补 rna 链到模板 rna 的酶。该酶不仅编码在病毒, 而且在模型有机体, 如植物, 酵母和蠕虫, 和双链 RNA 合成由 RdRP 贡献转录和转录后基因沉默在这些有机体5,6.虽然人类 RdRP 已经失踪很长一段时间, 我们发现 RdRP 活动的人叔在 2009年7。
首先用重组蛋白7证实了 RdRP 的活性, 然后建立了一种灵敏的体外检测方法, 测定内源性叔8的 RdRP 活性。在这里, 我们展示了体外RdRP 检测 (IP-RdRP 检测) 内源性叔。该方法始于内源性叔免疫沉淀 (IP), 其次是体外RdRP 反应, 其中放射性 ribonucleotides 被纳入新生 RNA 链。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Wako</td><td>044-29765</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>CORNING</td><td>35-010-CV</td><td></td></tr><tr><td>Penicillin-Streptomycin mixed solution</td><td>nacalai tesque</td><td>26253-84</td><td></td></tr><tr><td>Trypsin-Ethylenediamenetetraacetic acid (EDTA) solution</td><td>nacalai tesque</td><td>32777-44</td><td></td></tr><tr><td>Phosphate buffered saline (PBS(-))</td><td>Wako</td><td>166-23555</td><td></td></tr><tr><td>Thymidine</td><td>nacalai tesque</td><td>07147-61</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma</td><td>M1404</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>nacalai tesque</td><td>08904-85</td><td></td></tr><tr><td>Sodium chloride</td><td>nacalai tesque</td><td>31333-45</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>nacalai tesque</td><td>35434-21</td><td></td></tr><tr><td>Hydrochloric acid</td><td>nacalai tesque</td><td>18321-05</td><td></td></tr><tr><td>Nonidet P-40 (NP-40)</td><td>nacalai tesque</td><td>25223-04</td><td></td></tr><tr><td>Pierce Protein A Plus Agarose</td><td>Thermo scientific</td><td>22812</td><td></td></tr><tr><td>2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)</td><td>nacalai tesque</td><td>17514-15</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Wako</td><td>168-21815</td><td></td></tr><tr><td>Potassium acetate</td><td>nacalai tesque</td><td>28405-05</td><td></td></tr><tr><td>Magnesium chloride hexahydrate (MgCl2&amp;bull;6H2O)</td><td>Wako</td><td>135-00165</td><td></td></tr><tr><td>Glycerol</td><td>nacalai tesque</td><td>17045-65</td><td></td></tr><tr><td>Polyoxyethylene(10) octylphenyl ether (Triton X-100)</td><td>Wako</td><td>169-21105</td><td></td></tr><tr><td>cOmplete, EDTA-free</td><td>Roche Applied Science</td><td>11 873 580 001</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl2&amp;bull;2H2O)</td><td>nacalai tesque</td><td>06731-05</td><td></td></tr><tr><td>Micrococcal nuclease (MNase)</td><td>Takara Bio</td><td>2910A</td><td></td></tr><tr><td>Ethylene glycol bis (b-aminoethylether)-N,N,N&#039;,N&#039;-tetraacetic acid (EGTA)</td><td>nacalai tesque</td><td>15214-92</td><td></td></tr><tr><td>3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonat (CHAPS)</td><td>nacalai tesque</td><td>07957-64</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>nacalai tesque</td><td>14128-91</td><td></td></tr><tr><td>rCTP, rATP, rUTP, rGTP, 100mM each</td><td>Promega</td><td>E6000</td><td></td></tr><tr><td>[a-&lt;sup&gt;32&lt;/sup&gt;P]UTP (3,000 Ci/mmol)</td><td>PerkinElmer</td><td>NEG507H</td><td>Use fresh RI for the assay</td></tr><tr><td>RNase inhibitor</td><td>TOYOBO</td><td>SIN-201</td><td></td></tr><tr><td>Proteinase K</td><td>Takara</td><td>9033</td><td></td></tr><tr><td>Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td><td>Life Technologies</td><td>AM9720</td><td></td></tr><tr><td>3 M Sodium acetate</td><td>NIPPON GENE</td><td>316-90081</td><td></td></tr><tr><td>Ethanol (99.5)</td><td>nacalai tesque</td><td>14713-95</td><td></td></tr><tr><td>Dr. GenTLE Precipitation Carrier</td><td>Takara Bio</td><td>9094</td><td></td></tr><tr><td>RNase One Ribonuclease</td><td>Promega</td><td>M4265</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>nacalai tesque</td><td>02873-75</td><td></td></tr><tr><td>Formamide, deionized</td><td>nacalai tesque</td><td>16345-65</td><td></td></tr><tr><td>Ethylenediaminetetraacetic Acid Disodium Salt Dihydrate (EDTA)</td><td>nacalai tesque</td><td>15130-95</td><td></td></tr><tr><td>Orange G</td><td>nacalai tesque</td><td>25401-22</td><td></td></tr><tr><td>CO2 incubator</td><td>e.g., ASTEC</td><td>SCA-325DRS</td><td></td></tr><tr><td>Centrifuge</td><td>e.g., TOMY</td><td>EX-135</td><td>For step B)</td></tr><tr><td>Micro refrigerated centrifuge</td><td>e.g., KUBOTA</td><td>3740</td><td>For step 6.2</td></tr><tr><td>Sonicator</td><td>Qsonica</td><td>Q125</td><td>Set a microtip (#4422) on the sonicator, for step 6.4</td></tr><tr><td>Micro refrigerated centrifuge</td><td>e.g., TOMY</td><td>MX-305</td><td>For step 6.5</td></tr><tr><td>Cooled incubator</td><td>e.g., Panasonic</td><td>MIR-154-PJ</td><td>Set a rotary shaker inside</td></tr><tr><td>Rotary shaker</td><td>e.g., TAITEC</td><td>RT-5</td><td></td></tr><tr><td>Cooled incubator&amp;nbsp;</td><td>e.g., TAITEC</td><td>BR-43FL</td><td>Set a shaker &amp;ldquo;MINI WAVE&amp;rdquo; inside, for step 7.7</td></tr><tr><td>MINI WAVE</td><td>AS ONE</td><td>WEV-03</td><td>A shaker for steps 7.7, 8.5 and 8.6</td></tr><tr><td>Incubator</td><td>e.g., TAITEC</td><td>HB-80</td><td>Set a shaker &amp;ldquo;MINI WAVE&amp;rdquo; inside, for step 8.5 and 8.6</td></tr><tr><td>Block incubator</td><td>e.g., ASTEC</td><td>BI-516S</td><td></td></tr></tbody></table>

semi-quantitative detection of rna-dependent rna polymerase activity of human telomerase reverse transcriptase protein

端粒酶逆转录酶 (叔) 是端粒细胞的催化亚单位, 它通过 RNA 依赖性的 DNA 聚合酶活性拉长端粒。尽管叔被命名为逆转录酶, 但叔的结构和系统发育分析表明, 叔是右手聚合酶的成员, 与病毒 rna 相关的 rna 聚合酶 (RdRPs) 以及病毒逆转录酶有关。我们首先确定了人叔的 RdRP 活性, 产生互补 rna 立场的模板非编码 RNA, 并有助于 rna 沉默的癌细胞。为了分析这种非规范酶活性, 我们在2009年开发了重组叔 RdRP 化验, 然后建立了内源性叔体外RdRP 测定。在这篇手稿中, 我们描述后一种方法。简单地, 叔免疫复合物从细胞分离, 并与模板 RNA 和 rNTPs, 包括放射性 rNTP 的 RdRP 反应孵化。为消除单链 RNA, 用 RNase 对反应产物进行处理, 用聚丙烯酰胺凝胶电泳分析最终产物。核素 RdRP 产品可在隔夜曝光后显影检测到。

使用推荐的 RNA 模板, 放射性 RdRP 产物在20到30核苷酸 (nt) 之间被观察到, 特别是在有丝分裂阶段的 HeLa 细胞在过夜暴露以后 (图 1A)。通常, RdRP 产品的信号强度显示两个峰值, 它们位于 25 nt 和 30 nt 之间, 与下一代测序9确认的产品大小分布一致。与有丝分裂细胞相比, 没有同步的 HeLa 细胞几乎没有 20–30 nt 放射性产物。椭圆信号, 在所有样品被放置在 20 nt 以下, 是非特异性漂移。
如果在有丝分裂 HeLa 细胞中只有 30 nt 的产品有微弱信号, 则表明 RdRP 反应不顺利。例如, 如果 MNase 治疗是粗略和不恰当的, 有丝分裂 HeLa 细胞的信号强度显著下降 (图 1B)。RdRP 反应与旧的头盔的解决方案也减少了产品 (图 1C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57021/57021fig1.jpg"> 图 1: 在有丝分裂 HeLa 细胞中内源性叔 RdRP 的代表性产品。(A)三 nocodazole (操作) 或亚甲基亚砜 (unmanipulated) 治疗 HeLa 细胞中的 IP RdRP 测定结果。用化学合成的 34 nt RNA 作为模板。(B)在不适当的 MNase 治疗下进行的有丝分裂 HeLa 细胞的 RdRP 检测。(C)用新的或旧的显示器溶液进行的有丝分裂 HeLa 细胞的 RdRP 检测。<a href="https://cloudflare.jove.com/files/ftp_upload/57021/57021fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>试剂</td>
			<td>体积</td>
		</tr>
<tr>
<td>Micrococcal 核酸酶, 20 U/µL</td>
			<td>1µL</td>
		</tr>
<tr>
<td>Micrococcal 核酸酶缓冲器, 10x</td>
			<td>8µL</td>
		</tr>
<tr>
<td>RNase 水</td>
			<td>51µL</td>
		</tr>
</tbody></table>表 1: MNase 反应组合件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>试剂</td>
			<td>体积</td>
		</tr>
<tr>
<td>小伙子们, 0.07% (重量/音量)</td>
			<td>6µL</td>
		</tr>
<tr>
<td>头盔解决方案</td>
			<td>2µL</td>
		</tr>
<tr>
<td>rATP, 80 毫米</td>
			<td>0.5 µL</td>
		</tr>
<tr>
<td>rGTP, 8 毫米</td>
			<td>1µL</td>
		</tr>
<tr>
<td>rUTP, 0.4 毫米</td>
			<td>1µL</td>
		</tr>
<tr>
<td>rCTP, 8 毫米</td>
			<td>1µL</td>
		</tr>
<tr>
<td>RNase 抑制剂, 40 U/µL</td>
			<td>0.5 µL</td>
		</tr>
<tr>
<td>RNase 水</td>
			<td>1µL</td>
		</tr>
</tbody></table>表 2: RdRP 反应组合件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>试剂</td>
			<td>体积</td>
		</tr>
<tr>
<td>RNase 一核糖核酸, 10 U/µL</td>
			<td>0.2 µL</td>
		</tr>
<tr>
<td>反应缓冲器, 10x</td>
			<td>20µL</td>
		</tr>
<tr>
<td>RNase 水</td>
			<td>159.8 µL</td>
		</tr>
</tbody></table>表 3: 核糖核酸反应组合件。

Watch this Scientific Journal Video about Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein at JoVE.com

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

人端粒酶逆转录蛋白 rna 依赖性 rna 聚合酶活性的半定量检测

人端粒酶逆转录 (叔) 合成不仅端粒长度 DNA, 而且还通过 rna 依赖性 rna 聚合酶活性的双链 rna。在这里, 我们描述了一个新建立的检测 rna 依赖 rna 聚合酶活性的内源性叔。

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

semi-quantitative-detection-rna-dependent-rna-polymerase-activity

Research

JoVE Journal

Genetics

9.8K 次观看.  National Cancer Center Research Institute. 人端粒酶逆转录 (叔) 合成不仅端粒长度 DNA, 而且还通过 rna 依赖性 rna 聚合酶活性的双链 rna。在这里, 我们描述了一个新建立的检测 rna 依赖 rna 聚合酶活性的内源性叔。

视频: Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

科研

生物化学

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase chain reaction (PCR)-based method allows for robust measurements of enzyme activity from most cell lysates. The gel-based TRAP with fluorescently labeled primers limits sample throughput, and the ability to detect differences in samples is restricted to two fold or greater changes in enzyme activity. The droplet digital TRAP, ddTRAP, is a highly sensitive approach that has been modified from the traditional TRAP assay, enabling the user to perform a robust analysis on 96 samples per run and obtain absolute quantification of the DNA (telomerase extension products) input within each PCR. Therefore, the newly developed ddTRAP assay overcomes the limitations of the traditional gel-based TRAP assay and provides a more efficient, accurate, and quantitative approach to measuring telomerase activity within laboratory and clinical settings.

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.

液滴数字 TRAP (ddTRAP): 对端粒重复放大协议的适应性, 以实现液滴数字聚合酶链反应

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

癌症研究

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

常规 rt-pcr 和 sybr 绿色 rt-qpcr 检测罗非鱼

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

环境科学

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

RNA甲基转移酶活性分析的无抗体测定

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

遗传学

<meta charset="utf8"></head><body>人类群体研究中准确的端粒长度评估

Monochrome Multiplex Quantitative PCR Telomere Length Measurement

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. Telomere length (TL) has been associated with various exposures, biological processes, and health outcomes. This article describes the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay protocol routinely conducted in our laboratory for measuring relative mean TL from human DNA. There are several different PCR-based TL measurement methods, but the specific protocol for the MMqPCR method presented in this publication is repeatable, efficient, cost-effective, and suitable&#160;for population-based studies. This detailed protocol outlines all information necessary for investigators to establish this assay in their laboratory. In addition, this protocol provides specific steps to increase the reproducibility of TL measurement by this assay, defined by the intraclass correlation coefficient (ICC) across repeated measurements of the same sample. The ICC is a critical factor in evaluating expected power for a specific study population; as such, reporting cohort-specific ICCs for any TL assay is a necessary step to enhance the overall rigor of population-based studies of TL. Example results utilizing DNA samples extracted from peripheral blood mononuclear cells demonstrate the feasibility of generating highly repeatable TL data using this MMqPCR protocol.

<meta charset="utf8"></head><body>作者聚焦：探索创伤对细胞衰老的影响

Here we present a protocol for the measurement of relative telomere length (TL) using the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay. The MMqPCR assay is a repeatable, efficient, and cost-effective method for measuring TL from human DNA in population-based studies.

单色多重定量 PCR 端粒长度测量 

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. ...

使用非放射性化学发光检测定量端粒长度

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

作者聚焦：TAGGG 端粒长度测定性能参数的优化  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

TAGGG端粒长度测定性能参数优化

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

人端粒酶逆转录蛋白 rna 依赖性 rna 聚合酶活性的半定量检测

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此视频中的章节

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

液滴数字 TRAP (ddTRAP): 对端粒重复放大协议的适应性, 以实现液滴数字聚合酶链反应

常规 rt-pcr 和 sybr 绿色 rt-qpcr 检测罗非鱼

RNA甲基转移酶活性分析的无抗体测定

单色多重定量 PCR 端粒长度测量

单色多重定量 PCR 端粒长度测量

单色多重定量 PCR 端粒长度测量

TAGGG端粒长度测定性能参数优化

TAGGG端粒长度测定性能参数优化

TAGGG端粒长度测定性能参数优化