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 ...
[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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RNA Purification from the Unicellular Green Alga, Chromochloris zofingiensis
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Author:
Date:
2018-04-05
[Abstract] Chromochloris zofingiensis is a unicellular green alga that is an emerging model species for studies in fields such as biofuel production, ketocarotenoid biosynthesis and metabolism. The recent availability of a high-quality genome assembly facilitates systems-level analysis, such as RNA-Seq. However, cells of this alga have a tough cell wall, which is a challenge for RNA purification. This protocol was designed specifically to breach the cell wall and isolate high-quality RNA suitable for RNA-Seq studies.
[摘要] Chromochloris zofingiensis 是一种单细胞绿藻,是生物燃料生产,酮类胡萝卜素生物合成和代谢等领域研究的新兴模式物种。 最近获得的高质量基因组组装便于系统级分析,如RNA-Seq。 然而,这种藻类的细胞具有坚韧的细胞壁,这对于RNA纯化来说是一个挑战。 该方案专门设计用于破坏细胞壁并分离适合RNA-Seq研究的高质量RNA。
【背景】Chromochloris zofingiensis 是一种来自叶绿体谱系的小型单细胞绿藻(Dönz,1934)。此前,该物种在文献中已被描述为属于Muriella ,小球藻和 Mychonastes (Fučíková和Lewis,2012)。 C中有强烈的经济利益。因为它能够生产大量用于生物燃料的脂质,并且显示有望成为具有商业价值的营养制品虾青素的来源(Breuer等人,2012; Mulders等人。,2014; Liu et。,2016)。最近,发布了高质量的染色体水平的基因组组装和附带的注释,这有助于系统水平分析,如RNA-Seq(Roth等人,2017)。以下方案被设计为从液体培养物中产生高度纯化的总RNA,包括miRNA。 zofingiensis 适合RNA-Seq。 ℃。 zofingiensis 细胞受坚固细胞壁的保护,该细胞壁被设计为穿透。该方案的起始材料可以高达2.5×10 ...
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Detection and Analysis of Circular RNAs by RT-PCR
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Author:
Date:
2018-03-20
[Abstract] Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) ...
[摘要] 真核细胞中的基因表达在转录和转录后水平受到严格调控。 mRNA转录,mRNA转录和mRNA翻译等后转录过程由RNA结合蛋白(RBPs)和非编码(nc)RNAs控制。大量的ncRNA家族包含多种调控RNA,如microRNAs和长的非编码(lnc)RNAs,但也是探索不足的一类环状RNAs。虽然三十多年前电子显微镜首次发现,但只有高通量RNA测序(RNA-seq)的出现和创新生物信息学管道的开发已经开始允许系统鉴定circRNA(Szabo和Salzman,2016;熊猫,2017b;熊猫等,2017c)。然而,通过RNA测序鉴定的真正的circRNA的验证需要其他分子生物学技术,包括常规或定量(q)聚合酶链反应(PCR)和Northern印迹分析(Jeck和Sharpless,2014)的逆转录(RT)。使用不同引物的环状RNA的RT-qPCR分析已被广泛用于检测,验证和有时定量circRNA(Abdelmohsen等人,2015和2017; Panda等人, ,2017b)。如在此详述的,设计为跨越循环RNA后接连接序列的分歧引物可以特异性扩增circRNA而不是对应的线性RNA。总之,使用不同引物的RT-PCR分析允许直接检测和定量circRNA。
【背景】CircRNAs是共价闭合的,缺少5'或3'末端的单链RNA。虽然它们的起源知之甚少,但它们可以通过称为反向剪接的过程从前体mRNA产生(Panda等人,2017d; ...
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