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VeritiTM 96-Well Thermal Cycler

Veriti ® 96孔热循环仪

Company: Thermo Fisher Scientific
Catalog#: 4375786
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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; ...

A Small RNA Isolation and Sequencing Protocol and Its Application to Assay CRISPR RNA Biogenesis in Bacteria
Author:
Date:
2018-02-20
[Abstract]  Next generation high-throughput sequencing has enabled sensitive and unambiguous analysis of RNA populations in cells. Here, we describe a method for isolation and strand-specific sequencing of small RNA pools from bacteria that can be multiplexed to accommodate multiple biological samples in a single experiment. Small RNAs are isolated by polyacrylamide gel electrophoresis and treated with T4 polynucleotide kinase. This allows for 3’ adapter ligation to CRISPR RNAs, which don’t have pre-existing 3’-OH ends. Pre-adenylated adapters are then ligated using T4 RNA ligase 1 in the absence of ATP and with a high concentration of polyethylene glycol (PEG). The 3’ capture step enables precise determination of the 3’ ends of diverse RNA molecules. Additionally, a random hexamer in the ligated ... [摘要]  新一代高通量测序技术能够对细胞中的RNA群体进行敏感和明确的分析。在这里,我们描述了一种从细菌中分离和链特异性测序小RNA池的方法,所述细菌可以在单个实验中多路复用以容纳多个生物样品。小RNA通过聚丙烯酰胺凝胶电泳分离并用T4多核苷酸激酶处理。这允许3'衔接头连接至CRISPR RNA,其不具有预先存在的3'-OH末端。然后使用T4 RNA连接酶1在不存在ATP和高浓度聚乙二醇(PEG)的情况下将前腺苷酸化的衔接子连接。 3'捕获步骤能够精确测定不同RNA分子的3'末端。此外,连接适配器中的随机六聚体有助于控制潜在的下游扩增偏差。逆转录后,将cDNA产物环化并通过PCR制备文库。我们显示扩增的文库不需要通过凝胶电泳可见,以期望产物的有效测序。使用这种方法,我们通常从少量纯化的小RNA制备RNA测序文库。该协议适合于通过对成熟的CRISPR RNA进行测序来测定细菌中的CRISPR RNA生物合成,但可以用于测序不同类型的小RNA。我们还提供了一个完整的数据处理管道示例,并提供了运行所提供脚本的说明。


【背景】与聚集的经常散布的短回文重复序列(CRISPR)相关的遗传模块赋予不同的原核宿主适应性免疫(Barrangou et ...

Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii
Author:
Date:
2018-02-20
[Abstract]  Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii’s 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to functionally investigate hundreds of putative essential protein coding genes. Accordingly, we recently implemented the auxin-inducible degron (AID) system for studying essential proteins in T. gondii. Here we provide a step-by-step protocol for examining protein function in T. gondii using the AID system in a tissue culture setting. [摘要]  弓形虫是原生动物寄生虫称为Apicomplexa致命门的一员。 作为一个复杂的模型,关于T的信息有很多。 gondii的8,000多种蛋白质编码基因,包括序列变异,表达和对寄生虫适应的相对贡献。 然而,需要新的工具来功能性地调查数百个推定的必需蛋白质编码基因。 因此,我们最近实施了生长素诱导降解(AID)系统来研究T中的基本蛋白质。弓形虫。 在这里,我们提供了一个检查蛋白质功能的一步一步的协议。 在组织培养环境中使用AID系统。

【背景】生长素是一类通过靶向某些蛋白质在植物中进行蛋白酶体降解而发出信号的植物激素(Teale等人,2006)。 Kohei Nishimura等人具有将该植物特异性信号传导系统的组分转移到其他真核生物中用于有兴趣的蛋白质(POI)的条件调节,创建生长素诱导降解(AID)系统的聪明想法(Nishimura等人,2009)。这个系统已经被成功地用于几种真核生物,包括疟原虫疟原虫(Kreidenweiss et al。,2013; Philip和Waters,2015)。只需要两个转基因成分来实现这个系统,称为转运抑制剂反应1(TIR1)的植物生长素受体和用AID标记的POI。用生长素(例如,3-吲哚乙酸/ IAA)处理活化SCF ...

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