| Detecting Spaciotemporal Transcript Accumulation in Maize by RNA In Situ Hybridization
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Author:
Date:
2021-02-20
[Abstract] RNA in situ hybridization is a method for visualizing spatiotemporal transcript accumulation in cells and tissues. The method provides clear resolution, is highly sensitive and specific, and can uncover gradients of transcript accumulation within a histologically-intact tissue, which is not possible currently with other methods for transcript detection. RNA in situ hybridization, however, is not a quantitative approach for gene expression. Protocols for RNA in situ hybridization have numerous steps that can span several days of work, complicating troubleshooting procedures. Here, we build on previously published RNA in situ hybridization protocols optimized for paraffin-embedded and sectioned maize tissue (Jackson, 1991; Long et al., 1996 ; ...
[摘要] [摘要] RNA原位杂交是一种可视化时空转录本在细胞和组织中积累的方法。该方法提供清晰的分辨率，高度灵敏和特异，并且可以揭示组织完整的组织内转录物积累的梯度，这是当前其他方法无法检测到转录物的方法。然而，RNA原位杂交不是用于基因表达的定量方法。RNA原位杂交的协议有许多步骤，可能需要花费数天的时间，从而使故障排除过程变得更加复杂。在这里，我们基于先前发布的RNA原位构建 杂交方案针对石蜡包埋和切片的玉米组织进行了优化（Jackson，1991； Long等，1996；Javelle等，2011），它提供了优化转录本检测的其他措施。
图形摘要：
RNA原位杂交的工作流程
[背景技术]在时间和空间差异基因表达允许具有相同的遗传物质的细胞呈现不同的身份。确实，基因表达的这种变化通常负责驱动整个生物体中模式或形态的进化变化（Carroll，2005）。因此，通过观察组织内天然背景下的基因表达，可以增强对发育过程的见识。诸如RT-qPCR和RNA- ...
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| Charging State Analysis of Transfer RNA from an α-proteobacterium
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Author:
Date:
2020-12-05
[Abstract] Transfer RNA (tRNA) is an essential link between the genetic code and proteins. During the process of translation, tRNA is charged with its cognate amino acid and delivers it to the ribosome, thus serving as a substrate of protein synthesis. To analyze the charging state of a particular tRNA, total RNA is purified and analyzed on an acid-urea gel. Separated RNA is then transferred to a membrane and detected with a probe for the tRNA of interest. Here, we present an improved protocol to analyze the tRNA charging state in the α-proteobacterium Rhodopseudomonas palustris. Compared to the classical method, the RNA isolation step is optimized to suit this organism. Additionally, a non-radioactive platform is used for electrophoresis and Northern blots. This significantly reduces ...
[摘要] [摘要]转移RNA（tRNA）是遗传密码与蛋白质之间的重要纽带。在翻译过程中，tRNA带有其同源氨基酸，并将其传递至核糖体，因此可作为蛋白质合成的底物。为了分析特定tRNA的电荷状态，纯化总RNA并在酸性尿素凝胶上进行分析。然后将分离的RNA转移到膜上并用目标tRNA的探针进行检测。在这里，我们提出了一种改进的协议来分析α-变形杆菌Rhodopseudomonas palustris中的tRNA充电状态 。与传统方法相比，优化了RNA分离步骤以适合这种生物。另外，非放射性平台用于电泳和RNA印迹。这显着减少了此协议所需的时间和精力。
[背景] tRNA的主要功能是，与其他翻译因素的帮助，以确保mRNA的蛋白质的准确的翻译。氨基酰基-tRNA（带电）将氨基酸带到核糖体中以延长肽段，然后释放不带电荷的tRNA。tRNA的充电状态主要取决于可用资源（即氨基酸）及其被核糖体的消耗量。为了分析细胞tRNA的充电状态，已经开发了使用酸性脲凝胶分离总RNA并通过Northern印迹检测感兴趣的tRNA的方法（Janssen等人，2012； ...
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| Ribosome Purification from an α-proteobacterium and rRNA Analysis by Northern Blot
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Author:
Date:
2020-12-05
[Abstract] Ribosomes are an integral part of cellular life. They are complex molecular machines consisting of multiple ribosomal proteins and RNAs. To study different aspects of ribosome composition, many methods have been developed over the decades. Here, we describe how to purify ribosomes from the α-proteobacterium Rhodopseudomonas palustris. Following this protocol, RNA can be extracted from either purified ribosomes or directly from cell cultures, and ribosomal RNAs quantified using Northern blot. This protocol gives an example of studying ribosomes in a bacterium other than the commonly used E. coli. The challenge of performing Northern blots with rRNA is also addressed in detail.
[摘要] [摘要]核糖体是细胞生命的组成部分。它们是由多种核糖体蛋白和RNA组成的复杂分子机器。为了研究核糖体组成的不同方面，几十年来已经开发了许多方法。在这里，我们描述如何从α-变形杆菌Rhodopseudomonas palustris中纯化核糖体。按照该协议，可以从纯化的核糖体中提取RNA，也可以直接从细胞培养物中提取RNA，并使用Northern印迹对核糖体RNA进行定量。该协议给出了研究除常用大肠杆菌外的细菌中核糖体的一个例子。还详细介绍了使用rRNA进行Northern杂交的挑战。
[背景]细菌细胞的命运是紧密相连的核糖体。我们最近的研究表明，活性核糖体在营养缺乏的palustris细胞的存活机制中起着重要作用（Yin等人，2019）。核糖体通过一系列超速离心纯化，并采用经典方法优化的方法（Lawrence等，2016）。使用不太常用的毛细管转移系统，通过RNA印迹检测核糖体RNA群体。纯化步骤的细节可能极大地影响核糖体的状态。这些方法在这里进行了详细描述，对于研究多种细菌中的翻译设备的研究人员应该具有广泛的兴趣。
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