The stability of mRNAs is fundamental to determining expression level and dynamics. Nonetheless, current approaches for measuring transcript half-lives (e.g. transcription shutoff) are generally toxic or technically complex. Here we describe an alternative strategy for targeted measurements of endogenous mRNA stability that is simple, inexpensive, and non-toxic. Cells are first metabolically labelled with the nucleoside analog 4-thiouridine (4sU). Extracted mRNA can then be treated with the thiol-reactive compound N-ethylmaleimide. This compound modifies 4sU nucleotides and sterically interferes with reverse transcription of 4sU-containing transcripts, disrupting their conversion into cDNA. The decay rate of non-4sU-containing pre-existing mRNA can then be monitored by quantitative PCR (qPCR). Importantly, this approach avoids the biochemical isolation of 4sU-labelled transcripts and/or RNA-seq analysis required for other metabolic-labelling strategies. In summary, our method combines the simplicity of "transcription shutoff" strategies with the accuracy of metabolic-labelling strategies for measurements of mRNA stability across a wide range of half-lives.

中文翻译：

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Roadblock-qPCR：一种简单且廉价的 mRNA 稳定性靶向测量策略


mRNA 的稳定性对于确定表达水平和动态至关重要。尽管如此，目前测量转录半衰期（例如转录关闭）的方法通常是有毒的或技术复杂的。在这里，我们描述了一种简单、廉价且无毒的靶向测量内源 mRNA 稳定性的替代策略。首先用核苷类似物 4-硫尿苷 (4sU) 对细胞进行代谢标记。然后可以用硫醇反应性化合物 N-乙基马来酰亚胺处理提取的 mRNA。该化合物修饰 4sU 核苷酸，并在空间上干扰含有 4sU 的转录物的逆转录，破坏其向 cDNA 的转化。然后可以通过定量 PCR (qPCR) 监测不含 4sU 的预先存在的 mRNA 的衰减率。重要的是，这种方法避免了其他代谢标记策略所需的 4sU 标记转录本的生化分离和/或 RNA-seq 分析。总之，我们的方法将“转录关闭”策略的简单性与代谢标记策略的准确性结合起来，用于测量各种半衰期的 mRNA 稳定性。