| Measurement of mRNA Decay in Mouse Embryonic Fibroblasts
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Author:
Date:
2016-07-05
[Abstract] mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative transcript abundance using semi-quantitative real-time RT-PCR. Northern blotting or NanoString n-Counter are alternative methods to measure mRNA abundance, which is quantified using a phosphorimager in ...
[摘要] mRNA稳定性控制是基因表达的转录后调控中的关键步骤。放线菌素D，一种最初用作抗癌药物的抗生素，由于其对mRNA合成的抑制，已经证明是用于研究细胞中转录物的转换率的方便的工具。在这里，我们描述了添加放线菌素D后的稳定成纤维细胞细胞系，从野生型和三四氯丙胺（TTP）缺陷小鼠胚胎成纤维细胞（MEF）文化，以及一个协议使用半定量实时RT-PCR确定相对转录本丰度。 Northern印迹或NanoString n-Counter是测量mRNA丰度的备选方法，在前一种情况下使用phosphorimager对其进行定量。该方案适合于研究源自转基因小鼠及其各自对照的原代培养细胞和稳定细胞系，并且提供在具有和不具有目标基因的其他相同细胞中的mRNA衰减率的直接比较。
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| Extraction of Small RNA and qPCR Validation of miRNAs in Vigna mungo
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Author:
Date:
2015-03-05
[Abstract] Small RNAs like microRNAs (miRNAs), small interfering RNAs (siRNAs) and other noncoding RNAs including snRNA and snoRNA have tremendous impact on eukaryotic gene regulation. Extraction of high quality small RNAs is an important prerequisite for experimental analyses of miRNAs. This will prevent RNA degradation and remove associated contaminations including polyphenols, polysaccharides and other secondary metabolites. In this protocol we describe a simple way to isolate small RNAs from the leaf tissues of Vigna mungo combining the protocols of two commercially available kits with some modifications.
[摘要] 小RNA如微小RNA（miRNA），小干扰RNA（siRNA）和其他非编码RNA（包括snRNA和snoRNA）对真核基因调控具有巨大的影响。 高质量小RNA的提取是miRNA的实验分析的重要先决条件。 这将防止RNA降解和去除相关的污染物，包括多酚，多糖和其他次生代谢物。 在这个协议中，我们描述了一种简单的方法，从猕猴桃的叶组织中分离小RNAs，结合两个商业上可用的试剂盒的协议与一些修改。
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| Chromatin Immunoprecipitation (ChIP), Streptavidin and ATP-agarose Mediated Pull-down Analyses
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Author:
Date:
2013-09-20
[Abstract] Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) induces expression of both viral and cellular genes in virus infected B cells by mimicking activated Notch receptors (Notch-IC) that mediate transcription activation through binding to the repressing domain of the recombining binding protein suppressor of hairless (RBP-Jκ). In general, chromatin immunoprecipitation (ChIP) assays, electrophoresis mobility shift assays (EMSA), streptavidin-agarose mediated DNA pull-down assays, together with cell-based transcription reporter assays were conducted to verify whether the query protein is involved in EBNA2-dependent transcription. The ATP-bound state of nuclear chaperone nucleophosmin (NPM1) has been implicated in pleiotropic biological processes. An ATP-agarose-mediated pull-down protocol was ...
[摘要] EB病毒（EBV）核抗原2（EBNA2）通过模拟激活的Notch受体（Notch-IC）诱导病毒感染的B细胞中病毒和细胞基因的表达，所述Notch受体通过结合重组结合的抑制结构域介导转录激活无毛蛋白抑制剂（RBP-Jκ）。通常，进行染色质免疫沉淀（ChIP）测定，电泳迁移率变动测定（EMSA），链霉亲和素 - 琼脂糖介导的DNA下拉测定以及基于细胞的转录报道基因测定，以验证查询蛋白是否参与EBNA2依赖性转录。核伴侣核蛋白（NPM1）的ATP结合状态已涉及多效生物过程。开发ATP-琼脂糖介导的下拉方案以监测由ATP结合的NPM1诱导的引发前复合物的形成。根据EBNA2和Notch-IC已经显示出在B细胞系中靶基因的活化方面是部分可互换的，可以想象EBNA2是活化的Notch IC的生物学等价物。
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