来自活跃核糖体的记者新生链的免疫沉,以研究翻译效率
Roberta Cacioppo1, Catherine Lindon1
1Department of Pharmacology, University of Cambridge, Cambridge, UK.
Bio-protocol
|September 27, 2023
概括
我们开发了新生链免疫沉 (NC IP),这是一种简单的测试,用于测量特定mRNA的翻译效率. 这种方法使用免疫沉来隔离翻译核糖体,从而能够准确评估UTR,microRNA或药物对基因表达调节的作用.
科学领域:
- 分子生物学分子生物学
- 基因表达规范 基因表达规范
- 生物化学 生物化学
背景情况:
- 了解基因表达需要研究翻译效率 (TE).
- 测量TE的现有方法,如核糖体分析和记者测定,可能是复杂和劳动密集的.
- 需要更简单,更容易获得的方法来研究TE在个体基因水平.
研究的目的:
- 为研究mRNA翻译效率引入一种简单的生物化学记者试验.
- 提出新生链免疫沉 (NC IP) 作为一种分析从转染的等离子体中mRNA的TE的方法.
- 证明NC IP在研究UTR依赖的TE调节和区分翻译与非翻译mRNA中的实用性.
主要方法:
- 开发出新生链免疫沉 (NC IP),一种基于等离子体的测试.
- 利用从延长的核糖体中产生N-终端标记Flag的新生的多链的免疫沉.
- 通过定量逆转录聚合酶连锁反应 (RT-qPCR) 量化释放的mRNA,在释放后与氨酸或Flag一起.
主要成果:
- 该NC IP测定成功地测量了用户选择的mRNA的翻译效率.
- 使用NC IP方法证明了基于UTR的翻译效率调节.
- 该测试有效地区分了积极翻译的mRNA与非翻译的mRNA.
结论:
- NC IP提供了一种简单,高效和基于标准实验室设备的方法来研究mRNA翻译.
- 该技术可用于研究各种因素,包括微RNA,药物和遗传背景对TE的影响.
- 该NC IP协议可适应在不同的生物体中使用,例如哺乳动物细胞和芽酵母.
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