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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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