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RNeasy Mini Kit (50)

RNeasy迷你套件

Company: QIAGEN
Catalog#: 74104
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Generation of Human iPSC-derived Neural Progenitor Cells (NPCs) as Drug Discovery Model for Neurological and Mitochondrial Disorders
Author:
Date:
2021-03-05
[Abstract]  

The high attrition rate in drug development processes calls for additional human-based model systems. However, in the context of brain disorders, sampling live neuronal cells for compound testing is not applicable. The use of human induced pluripotent stem cells (iPSCs) has revolutionized the field of neuronal disease modeling and drug discovery. Thanks to the development of iPSC-based neuronal differentiation protocols, including tridimensional cerebral organoids, it is now possible to molecularly dissect human neuronal development and human brain disease pathogenesis in a dish. These approaches may allow dissecting patient-specific treatment efficacy in a disease-relevant cellular context. For drug discovery approaches, however, a highly reproducible and cost-effective cell model is

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[摘要]  [摘要]药物开发过程中的高流失率要求使用其他基于人的模型系统。但是,在脑部疾病的情况下,不适合对活的神经元细胞进行采样以进行化合物测试。人类诱导的多能干细胞(iPSC )的使用彻底改变了神经元疾病建模和药物发现领域。由于基于iPSC的神经元分化方案(包括三维脑类器官)的发展,现在可以在一个碟子中分子解剖人神经元发育和人脑疾病的发病机理。这些方法可以允许在与疾病相关的细胞环境中解剖患者特异性的治疗功效。但是,对于药物发现方法,需要高度可复制且具有成本效益的细胞模型。在这里,我们描述了一种一步-步骤,用于从人产生健壮和可膨胀的神经祖细胞(NPC)工艺的iPSC 。用此协议生成的NPC是同质的且高度增殖。这些功能使NPC适合开发用于药物发现的高通量化合物筛选。人iPSC衍生的NPC示出了代谢依赖于线粒体活性,因此可也用于研究神经病症,其中线粒体功能受到影响。该协议涵盖了制备,培养和表征人iPSC来源的NPC所需的所有步骤。


图形摘要:


示意性的协议的所述发电机密封的离子人类源自iPSC的的NPC

[背景技术]近年来,目标为中心的药物发现的缺点已经用于寻址的神经系统疾病的方案变得明显,特别是(保罗等人,2010) ...

Identification of Intrinsic RNA Binding Specificity of Purified Proteins by in vitro RNA Immunoprecipitation (vitRIP)
Author:
Date:
2021-03-05
[Abstract]  

RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target selectivity and function of an individual protein.


The presented in vitro RNA immunoprecipitation assay (vitRIP) uncovers intrinsic RNA binding specificities of isolated proteins using the total cellular RNA pool as a library. Total RNA extracted from cells or tissues is incubated with purified recombinant proteins, RNA-protein complexes are immunoprecipitated and bound transcripts are identified by deep sequencing or quantitative RT-PCR.

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[摘要]  [摘要] RNA-蛋白质相互作用通常由专门的规范RNA结合域介导。然而,越来越多地观察到通过具有未知特异性的非经典结构域的相互作用,这提出了如何识别RNA靶标的问题。内在的RNA结合特异性的知识有助于理解单个蛋白质的靶标选择性和功能。

所呈现的体外RNA免疫沉淀测定法(vitRIP )揭示固有RNA使用总细胞RNA池作为分离的蛋白质的结合特异性一个库。从细胞或组织中提取的总RNA与纯化的重组蛋白孵育,免疫沉淀RNA-蛋白复合物,并通过深度测序或定量RT-PCR鉴定结合的转录物。这些RNA中丰富的RNA类和核苷酸频率决定了重组蛋白的固有特异性。该简单而通用的方案可适用于任何细胞类型或组织的其他RNA结合蛋白和总RNA文库。



图形摘要:


图1.体外RNA免疫沉淀(vitRIP )方案示意图

[背景]真核细胞包含许多不同的RNA类,具有成千上万的RNA种类以及与之相互作用的高度多样化的蛋白质。根据结合的RNA序列或结构的定义以及相互作用中涉及的蛋白质结构域的不同,RNA-蛋白质相互作用可分为特异性和非特异性(Jankowsky和Harris,2015)。越来越多地观察到通过未知特异性的非经典RNA结合结构域进行的RNA相互作用,这提出了如何识别专用RNA靶标的问题。 ...

Candida albicans Culture, Cell Harvesting, and Total RNA Extraction
Author:
Date:
2020-11-05
[Abstract]  

Transcriptional analysis has become a cornerstone of biological research, and with the advent of cheaper and more efficient sequencing technology over the last decade, there exists a need for high-yield and efficient RNA extraction techniques. Fungi such as the human pathogen Candida albicans present a unique obstacle to RNA purification in the form of the tough cell wall made up of many different components such as chitin that are resistant to many common mammalian or bacterial cell lysis methods. Typical in vitro C. albicans cell harvesting methods can be time consuming and expensive if many samples are being processed with multiple opportunities for product loss or sample variation. Harvesting cells via vacuum filtration rather than centrifugation cuts down on time before the cells are

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[摘要]  [摘要]转录分析已成为生物学研究的基石,并且在最近十年中,随着廉价,高效测序技术的出现,对高产量,高效率的RNA提取技术存在着需求。真菌如人病原体白色念珠菌呈现出独特的障碍RNA纯化在坚韧细胞壁的形式作出许多不同的部件,例如几丁质的最多的是对许多常见的哺乳动物细胞或细菌细胞裂解的方法具有抗性。典型的体外白色念珠菌 如果要处理的样品很多,并且产品损失或样品变化的机会很多,则细胞采集方法可能既耗时又昂贵。通过真空过滤而不是离心收集细胞可以减少冷冻前的时间,因此可以改变RNA表达谱的可用时间。对于白色念珠菌而言,真空过滤是优选的,这主要有两个原因:由于暴露的表面积增加,非丸状细胞的细胞裂解速度更快,并且与酵母或细菌细胞不同,丝状细胞首先难以沉淀。与酶法处理相比,使用机械细胞裂解法(通过氧化锆/二氧化硅珠子)可减少加工时间以及总成本。总的来说,该方法是从白念珠菌的体外培养物中提取总RNA的快速,高效且高产率的方法。

[背景]需要快速,可重复的,高效的RNA提取技术已经显著在过去几年里,由于使用RNA测序等表达分析技术的稳步增长成长是变得更加经济实惠,并与测序技术的改进速度更快。各种公司和实验室提供了许多不同的工具包和协议,试图满足这一需求。但是,专门为一种真菌建立的方法可能不适用于另一种真菌,而试剂盒平台由于其应用范围太广,常常会不足。在这里,我们描述了一种细胞培养物,收获,和RNA提取方法为致病性真菌,白色念珠菌,其利用的技术的组合,得到以一致和有效的方式既高产率和高质量的RNA。 ...

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