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dNTP Mix (10 mM each)

dNTP Mix (10 mM each)

Company: Thermo Fisher Scientific
Catalog#: R0193
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Detection and Analysis of Circular RNAs by RT-PCR
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) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al., 2017b; Panda et al., ... [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; ...

Polysome Fractionation to Analyze mRNA Distribution Profiles
Author:
Date:
2017-02-05
[Abstract]  Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ... [摘要]  真核细胞通过精确调节特定基因产物的表达来适应外部或内部信号的变化。蛋白质编码基因的表达受到转录和转录后水平的控制。在后面的步骤中,翻译的调节在需要蛋白质表达模式快速变化的细胞过程中特别重要。 mRNA的翻译效率由RNA结合蛋白(RBP)和非编码(nc)RNA如微RNA(Panda等人,2014a和2014b; Abdelmohsen等人)改变2014)。调节选择性mRNA翻译的因素的影响是RNA生物学中的一个关键问题。多核糖体(多核糖体)分馏分析是评估核糖体与给定mRNA的关联的有效方法。它提供了关于该mRNA的翻译状态的有价值的信息,这取决于与它们相关联的核糖体的数目,并且鉴定未翻译的mRNA(Panda等人,2016)。与许多核糖体相关的mRNA形成大量的多核糖体,预计将被主动翻译,而与少数或没有核糖体相关的mRNA有可能翻译不佳。总之,多聚糖分馏分析允许直接测定整个转录组水平的翻译效率以及个体mRNA。

背景 ...

Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes
Author:
Date:
2016-12-20
[Abstract]  RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al., 2008). There are several methodologies available today to identify RNAs bound to specific RBPs; some detect only recombinant molecules in vitro, others detect recombinant and endogenous molecules, while others detect only endogenous molecules. Examples include systematic evolution of ligands by exponential enrichment (SELEX), biotinylated RNA pulldown assay, RNA immunoprecipitation (RIP) assay, electrophoretic mobility shift assay ... [摘要]  RNA结合蛋白(RBP)近来已经成为调控基因表达的关键因素。 RBP与靶mRNA的相互作用通过改变不同的调节步骤来控制基因产物的水平,包括mRNA前体剪接和成熟,核mRNA输出和mRNA稳定性和翻译(Glisovic et al。,2008) )。目前有几种方法可用于鉴定与特定RBP结合的RNA;一些仅在体外检测重组分子,其他检测重组和内源性分子,而其他检测仅内源性分子。实例包括通过指数富集(SELEX),生物素化RNA下拉测定,RNA免疫沉淀(RIP)测定,电泳迁移率变动测定(EMSA),RNA足迹分析和各种UV交联和免疫沉淀(CLIP)方法如CLIP ,PAR-CLIP和iCLIP(Popova等人,2015)。在这里,我们描述了一种简单而有信息的方法来研究和鉴定RBP与其目标转录物之间相互作用的RNA区域(Panda等人,2014和2016)。其重现性和易用性使得该方案成为识别RBP与特异性RNA之间相互作用的快速有效的方法。

背景 RNA蛋白相互作用严重影响基因表达模式。这些核糖核蛋白(RNP)复合物的鉴定对于理解由RNA结合蛋白(RBP)控制的调控机制是必不可少的。最近,广泛的努力导致了开发用于系统分析RNA-蛋白质相互作用的方法。识别RNP复合物的高度信息化方法包括许多不同类型的RNP免疫沉淀(IP)分析。 ...

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