| Molecular and Phenotypic Characterization Following RNAi Mediated Knockdown in Drosophila
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Author:
Date:
2021-02-20
[Abstract] Loss of function studies shed significant light on the involvement of a gene or gene product in different cellular processes. Short hairpin RNA (shRNA) mediated RNA interference (RNAi) is a classical yet straightforward technique frequently used to knock down a gene for assessing its function. Similar perturbations in gene expression can be achieved by siRNA, microRNA, or CRISPR-Cas9 methods also. In Drosophila genetics, the UAS-GAL4 system is utilized to express RNAi and make ubiquitous and tissue-specific knockdowns possible. The UAS-GAL4 system borrows genetic components of S. cerevisiae, hence rule out the possibility of accidental expression of the system. In particular, this technique uses a target-specific shRNA, and the expression of the same is governed by the upstream activating ...
[摘要] [摘要]功能丧失的研究为基因或基因产物在不同细胞过程中的参与提供了重要启示。短发夹RNA（shRNA）介导的RNA干扰（RNAi）是一种经典而直接的技术，经常用于敲低基因以评估其功能。也可以通过siRNA，microRNA或CRISPR-Cas9方法实现类似的基因表达扰动。在果蝇遗传学中，UAS-GAL4系统用于表达RNAi，并使遍在和组织特异性的基因敲除成为可能。UAS-GAL4系统借鉴了酿酒酵母的遗传成分，因此排除了系统意外表达的可能性。特别地，该技术使用靶标特异性shRNA，并且其表达受上游激活序列（UAS）支配。由特定启动子调节的GAL4受控表达可以普遍或以组织特异性方式驱动干扰RNA的表达。通过RNA分离和半定量RT-PCR反应，然后进行琼脂糖凝胶电泳来测量敲低效率。我们还采用了免疫染色程序来评估击倒效率。
RNAi为研究人员提供了降低基因产物水平（相当于亚同型条件）并研究结果的选择。基于UAS-GAL4的RNAi方法提供了基因表达的时空调节，还有助于推断早期发育阶段所需的基因功能。
[背景]果蝇果蝇（果蝇）是在研究实验室经常使用的一种通用模式生物。果蝇易于处理，繁殖和维护。而且，精心制作却寿命短，繁殖力高的果蝇具有更多的优势。果蝇遗传学工具的易用性有助于发展对基因功能的全面了解。由于果蝇基因中有60％与人类基因同源，并且具有前面提到的其他优点，因此果蝇是研究体内基因功能的显而易见的模型生物。 ...
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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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| Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue
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Author:
Date:
2020-10-20
[Abstract] Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from ...
[摘要] [摘要] 转录组分析可以为不同遗传背景或不同条件下的生物学过程提供线索。RNA测序（RNA-seq）的价格已经下降到足够低的程度，因此在各种条件下进行中大规模转录组分析是可行的。然而，对于那些希望第一次使用RNA-seq或进行单个实验的人来说，RNA-seq库制备的价格和各种选择仍然是令人望而生畏的。选择文库制备方案的标准包括RNA分离方法、核苷酸片段化以获得所需的大小范围，以及文库索引以汇集测序样本进行多路复用。在这里，我们提出了一个高质量和高通量的选择，从多聚腺苷酸mRNA制备文库用于转录组分析。高质量和高通量的方案选择都包括通过磁珠使poly-A尾部沉淀，cDNA合成，然后通过Tn5介导的“标记”同时裂解和添加适配器的步骤。该方案的所有步骤均已通过拟南芥叶片和幼苗组织的验证，并简化为协同工作，在资金和时间上成本最低，因此旨在为转录组分析提供一个初学者友好的从开始到完成的RNA序列库制备。
[背景] 通过Southern印迹、expressed sequence ...
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