| RNase Sensitivity Screening for Nuclear Bodies with RNA Scaffolds in Mammalian Cells
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Author:
Date:
2017-04-20
[Abstract] The mammalian cell nucleus is highly organized and contains membraneless nuclear bodies (NBs) characterized by distinct resident factors. The NBs are thought to serve as sites for biogenesis and storage of certain RNA and protein factors as well as assembly of ribonucleoprotein complexes. Some NBs are formed with architectural RNAs (arcRNAs) as their structural scaffolds and additional NBs likely remain unidentified in mammalian cells. Here, we describe an experimental protocol to search for new NBs built on certain arcRNAs. RNase-sensitive NBs were identified by monitoring nuclear foci visualized by tagging thousands of human cDNA products.
[摘要] 哺乳动物细胞核高度组织，包含以不同的居民因素为特征的无膜核体（NBs）。 NB被认为是用于某些RNA和蛋白质因子的生物发生和储存的位点以及核糖核蛋白复合物的组装。 一些NB由构建的RNA（arcRNA）形成，作为它们的结构性支架，另外的NB可能在哺乳动物细胞中保持不明。 在这里，我们描述了一个实验协议来搜索建立在某些arcRNA上的新NB。 通过监测通过标记数千个人类cDNA产物可视化的核病灶来鉴定RNase敏感性NB。
【背景】哺乳动物细胞核是高度组织的，由称为核体（NB）的多个不同的亚结构组成。迄今为止，已经将15个NB鉴定为含有各种蛋白质和RNA因子的亚核膜无颗粒结构，其中许多颗粒结构用作特异性RNA，蛋白质和/或核糖核蛋白（RNP）复合物的生物发生，成熟，储存和螯合的位点Mao et al。，2011; Sleeman and Trinkle-Mulcahy，2014）（表1）。
一些NB被构建在称为结构RNA（arcRNA）的特定长非编码RNA（lncRNA）上，其定义为NB的结构核心（Chujo等，2016）。 arcRNA依赖的NB由与arcRNA相互作用的许多RNA结合蛋白组成。最显着的例子是由几种特征性RNA结合蛋白组成的寄生虫斑（Fox等，2002; ...
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| A SYBR Green-based Real Time RT-PCR Assay for Detection of the Emerging H7N9 Virus
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Author:
Date:
2014-06-20
[Abstract] Most recently a novel avian-origin influenza A (H7N9) virus emerged in China and has been associated with lots of human infection and fatal cases. Molecular diagnostic methods are thus urgently needed in public health laboratories. We developed a SYBR green-based one-step real time reverse transcription-PCR (RT-PCR) to detect the novel H7N9 virus.
[摘要] 最近，一种新型禽流感A型（H7N9）病毒在中国出现，并且与许多人类感染和致命病例有关。 因此，公共卫生实验室迫切需要分子诊断方法。 我们开发了基于SYBR绿色的一步实时逆转录PCR（RT-PCR），以检测新型H7N9病毒。
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| Isolation of Radiolabeled Poliovirus Particles from H1 HeLa Cells
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Author:
Date:
2013-08-20
[Abstract] The following protocol describes the isolation of radioactive viral and subviral particles from infected cells. This protocol has been written for isolation of poliovirus particles from H1 HeLa cells. Infection protocol and timing of [35S] methionine labeling and particle collection should be tailored to the virus of interest. Following isolation of the viral particles, the viral proteins present in these particles may be separated by gel electrophoresis and visualized by autoradiography.
[摘要] 以下方案描述了从感染的细胞中分离放射性病毒和亚病毒颗粒。 该协议是为了从H1 HeLa细胞分离脊髓灰质炎病毒颗粒而编写的。 [35]甲硫氨酸标记和颗粒收集的感染方案和时机应该针对感兴趣的病毒定制。 分离病毒颗粒后，可以通过凝胶电泳分离存在于这些颗粒中的病毒蛋白，并通过放射自显影进行显现。
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