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>

<ol>
	<li>Martinez, P., Blasco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+and+extra-telomeric+roles+for+telomerase+and+the+telomere-binding+proteins.">Telomeric and extra-telomeric roles for telomerase and the telomere-binding proteins.</a> Nat Rev Cancer. 11 (3), 161-176 (2011).</li><li>Nakamura, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+catalytic+subunit+homologs+from+fission+yeast+and+human.">Telomerase catalytic subunit homologs from fission yeast and human.</a> Science. 277 (5328), 955-959 (1997).</li><li>Gillis, A. J., Schuller, A. P., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+Tribolium+castaneum+telomerase+catalytic+subunit+TERT.">Structure of the Tribolium castaneum telomerase catalytic subunit TERT.</a> Nature. 455 (7213), 633-637 (2008).</li><li>Salgado, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+an+RNAi+polymerase+links+RNA+silencing+and+transcription.">The structure of an RNAi polymerase links RNA silencing and transcription.</a> PLoS Biol. 4 (12), e434 (2006).</li><li>Castel, S. E., Martienssen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+the+nucleus:+roles+for+small+RNAs+in+transcription,+epigenetics+and+beyond.">RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond.</a> Nat Rev Genet. 14 (2), 100-112 (2013).</li><li>Sugiyama, T., Cam, H., Verdel, A., Moazed, D., Grewal, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-dependent+RNA+polymerase+is+an+essential+component+of+a+self-enforcing+loop+coupling+heterochromatin+assembly+to+siRNA+production.">RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production.</a> Proc Natl Acad Sci U S A. 102 (1), 152-157 (2005).</li><li>Maida, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA-dependent+RNA+polymerase+formed+by+TERT+and+the+RMRP+RNA.">An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA.</a> Nature. 461 (7261), 230-235 (2009).</li><li>Maida, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+telomerase+reverse+transcriptase+in+heterochromatin+maintenance.">Involvement of telomerase reverse transcriptase in heterochromatin maintenance.</a> Mol Cell Biol. 34 (9), 1576-1593 (2014).</li><li>Maida, Y., Yasukawa, M., Masutomi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+Novo+RNA+Synthesis+by+RNA-Dependent+RNA+Polymerase+Activity+of+Telomerase+Reverse+Transcriptase.">De Novo RNA Synthesis by RNA-Dependent RNA Polymerase Activity of Telomerase Reverse Transcriptase.</a> Mol Cell Biol. 36 (8), 1248-1259 (2016).</li><li>Okamoto, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+tumor+initiating+cells+of+defined+genetic+composition+by+nucleostemin.">Maintenance of tumor initiating cells of defined genetic composition by nucleostemin.</a> Proc Natl Acad Sci U S A. 108 (51), 20388-20393 (2011).</li><li>van Dijk, A. A., Makeyev, E. V., Bamford, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+viral+RNA-dependent+RNA+polymerization.">Initiation of viral RNA-dependent RNA polymerization.</a> J Gen Virol. 85 (Pt 5), 1077-1093 (2004).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1082">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:464px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:155px;">Wako</td>
			<td style="width:119px;">044-29765</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum (FBS)</td>
			<td>CORNING</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin mixed solution</td>
			<td>nacalai tesque</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-Ethylenediamenetetraacetic acid (EDTA) solution</td>
			<td>nacalai tesque</td>
			<td>32777-44</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline (PBS(-))</td>
			<td>Wako</td>
			<td>166-23555</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thymidine</td>
			<td>nacalai tesque</td>
			<td>07147-61</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nocodazole</td>
			<td>Sigma</td>
			<td>M1404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>nacalai tesque</td>
			<td>08904-85</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>nacalai tesque</td>
			<td>31333-45</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris(hydroxymethyl)aminomethane</td>
			<td>nacalai tesque</td>
			<td>35434-21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid</td>
			<td>nacalai tesque</td>
			<td>18321-05</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nonidet P-40 (NP-40)</td>
			<td>nacalai tesque</td>
			<td>25223-04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Protein A Plus Agarose</td>
			<td>Thermo scientific</td>
			<td align="right">22812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)</td>
			<td>nacalai tesque</td>
			<td>17514-15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium hydroxide</td>
			<td>Wako</td>
			<td>168-21815</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium acetate</td>
			<td>nacalai tesque</td>
			<td>28405-05</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate (MgCl2&#8226;6H2O)</td>
			<td>Wako</td>
			<td>135-00165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>nacalai tesque</td>
			<td>17045-65</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyoxyethylene(10) octylphenyl ether (Triton X-100)</td>
			<td>Wako</td>
			<td>169-21105</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cOmplete, EDTA-free</td>
			<td>Roche Applied Science</td>
			<td>11 873 580 001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate (CaCl2&#8226;2H2O)</td>
			<td>nacalai tesque</td>
			<td>06731-05</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micrococcal nuclease (MNase)</td>
			<td>Takara Bio</td>
			<td>2910A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylene glycol bis (b-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td>nacalai tesque</td>
			<td>15214-92</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonat (CHAPS)</td>
			<td>nacalai tesque</td>
			<td>07957-64</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td>nacalai tesque</td>
			<td>14128-91</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rCTP, rATP, rUTP, rGTP, 100mM each</td>
			<td>Promega</td>
			<td>E6000</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">[a-32P]UTP (3,000 Ci/mmol)</td>
			<td>PerkinElmer</td>
			<td>NEG507H</td>
			<td>Use fresh RI for the assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase inhibitor</td>
			<td>TOYOBO</td>
			<td>SIN-201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Proteinase K</td>
			<td>Takara</td>
			<td align="right">9033</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td>
			<td>Life Technologies</td>
			<td>AM9720</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3 M Sodium acetate</td>
			<td>NIPPON GENE</td>
			<td>316-90081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol (99.5)</td>
			<td>nacalai tesque</td>
			<td>14713-95</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dr. GenTLE Precipitation Carrier</td>
			<td>Takara Bio</td>
			<td align="right">9094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase One Ribonuclease</td>
			<td>Promega</td>
			<td>M4265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecyl sulfate (SDS)</td>
			<td>nacalai tesque</td>
			<td>02873-75</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formamide, deionized</td>
			<td>nacalai tesque</td>
			<td>16345-65</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminetetraacetic Acid Disodium Salt Dihydrate (EDTA)</td>
			<td>nacalai tesque</td>
			<td>15130-95</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orange G</td>
			<td>nacalai tesque</td>
			<td>25401-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CO2 incubator</td>
			<td>e.g., ASTEC</td>
			<td>SCA-325DRS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>e.g., TOMY</td>
			<td>EX-135</td>
			<td>For step B)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro refrigerated centrifuge</td>
			<td>e.g., KUBOTA</td>
			<td align="right">3740</td>
			<td>For step 6.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator</td>
			<td>Qsonica</td>
			<td>Q125</td>
			<td>Set a microtip (#4422) on the sonicator, for step 6.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro refrigerated centrifuge</td>
			<td>e.g., TOMY</td>
			<td>MX-305</td>
			<td>For step 6.5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cooled incubator</td>
			<td>e.g., Panasonic</td>
			<td>MIR-154-PJ</td>
			<td>Set a rotary shaker inside</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotary shaker</td>
			<td>e.g., TAITEC</td>
			<td>RT-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cooled incubator&#160;</td>
			<td>e.g., TAITEC</td>
			<td>BR-43FL</td>
			<td>Set a shaker &#8220;MINI WAVE&#8221; inside, for step 7.7</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>e.g., TAITEC</td>
			<td>HB-80</td>
			<td>Set a shaker &#8220;MINI WAVE&#8221; inside, for step 8.5 and 8.6</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Genetics

9.7K 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

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

可诱导T7 RNA聚合酶介导的多基因表达系统，pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

科研

遗传学

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

酿酒酵母4-硫尿嘧啶代谢标记及新合成 mRNA 作为 RNA 聚合酶 II. 活性的一种代理

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

定量细菌中蛋白质-RNA结合的测定

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

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 ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

人类胰腺胰岛的单细胞RNA测序与分析

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...