Suppr超能文献

RNA翻译的靶向分析

Targeted Profiling of RNA Translation.

作者信息

Li Ben B, Qian Changli, Roberts Thomas M, Zhao Jean J

机构信息

Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts.

出版信息

Curr Protoc Mol Biol. 2019 Jan;125(1):e71. doi: 10.1002/cpmb.71. Epub 2018 Oct 22.

DOI:10.1002/cpmb.71
PMID:30346115
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6326074/
Abstract

This unit describes a reverse transcription-quantitative PCR (RT-qPCR)-based method for gene-targeted measurement of RNA translation levels. The method includes washing and lysing cells with a buffer containing cycloheximide to enrich ribosomal accumulation at translation initiation sites (TIS), followed by enzymatic treatment to generate ribosomal footprints, reverse transcription targeted towards TIS of specific transcripts of interest to generate complementary DNA (cDNA), and qPCR to measure the abundance of these footprints. This method enables time- and cost-effective assessment of changes in translation levels across focused panels of genes and across numerous samples. © 2018 by John Wiley & Sons, Inc.

摘要

本单元介绍了一种基于逆转录定量PCR（RT-qPCR）的方法，用于基因靶向测量RNA翻译水平。该方法包括用含有环己酰亚胺的缓冲液洗涤和裂解细胞，以富集翻译起始位点（TIS）处的核糖体积累，随后进行酶处理以产生核糖体足迹，针对感兴趣的特定转录本的TIS进行逆转录以生成互补DNA（cDNA），并通过qPCR测量这些足迹的丰度。该方法能够在时间和成本效益方面评估跨聚焦基因面板和众多样本的翻译水平变化。© 2018约翰威立国际出版公司。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fa2e/6326074/85b76c1e41a8/nihms-989677-f0001.jpg

相似文献

1
Targeted Profiling of RNA Translation.RNA翻译的靶向分析
Curr Protoc Mol Biol. 2019 Jan;125(1):e71. doi: 10.1002/cpmb.71. Epub 2018 Oct 22.
2
Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples.基于FFPE样本的简单且灵敏的逆转录定量聚合酶链反应基因表达分析
Sci Rep. 2016 Feb 22;6:21418. doi: 10.1038/srep21418.
3
Monitoring gene expression: quantitative real-time rt-PCR.监测基因表达：定量实时逆转录聚合酶链反应
Methods Mol Biol. 2013;1027:19-45. doi: 10.1007/978-1-60327-369-5_2.
4
RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells.RNA扩增方案导致聚合酶链反应结果出现偏差，尤其是对于人骨髓来源基质细胞的低拷贝转录本。
PLoS One. 2015 Oct 20;10(10):e0141070. doi: 10.1371/journal.pone.0141070. eCollection 2015.
5
RT-qPCR gene expression analysis in zebrafish: Preanalytical precautions and use of expressed repetitive elements for normalization.斑马鱼中的RT-qPCR基因表达分析：分析前的注意事项以及使用表达的重复元件进行标准化
Methods Cell Biol. 2016;135:329-42. doi: 10.1016/bs.mcb.2016.02.002. Epub 2016 Mar 4.
6
Bayesian modeling of reproducibility and robustness of RNA reverse transcription and quantitative real-time polymerase chain reaction.贝叶斯建模法在 RNA 逆转录和实时定量聚合酶链反应重现性和稳健性中的应用。
Anal Biochem. 2012 Sep 1;428(1):81-91. doi: 10.1016/j.ab.2012.06.010. Epub 2012 Jun 17.
7
Reverse Transcription-Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) for Gene Expression Analyses.逆转录-定量实时聚合酶链反应 (RT-qPCR) 用于基因表达分析。
Methods Mol Biol. 2022;2508:319-340. doi: 10.1007/978-1-0716-2376-3_21.
8
High-Throughput Plant Gene Expression Analysis by 384-Format Reverse Transcription-Quantitative PCR for Investigating Plant Iron Homeostasis.采用384孔板逆转录定量PCR进行高通量植物基因表达分析以研究植物铁稳态
Methods Mol Biol. 2023;2665:1-22. doi: 10.1007/978-1-0716-3183-6_1.
9
Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression.用于分析金黄色葡萄球菌基因表达的定量实时PCR（qPCR）工作流程。
Methods Mol Biol. 2016;1373:143-54. doi: 10.1007/7651_2014_193.
10
Gene expression analysis in biomarker research and early drug development using function tested reverse transcription quantitative real-time PCR assays.利用功能验证的反转录实时定量 PCR 分析方法进行生物标志物研究和早期药物开发中的基因表达分析。
Methods. 2013 Jan;59(1):10-9. doi: 10.1016/j.ymeth.2012.07.003. Epub 2012 Jul 14.

引用本文的文献

1
PPP6R3-mediated dephosphorylation regulates mRNA translation during spermatogonial differentiation.PPP6R3介导的去磷酸化在精原细胞分化过程中调节mRNA翻译。
Commun Biol. 2025 Jul 28;8(1):1114. doi: 10.1038/s42003-025-08539-1.
2
The ribosomal chaperone NACA recruits PHD2 to cotranslationally modify HIF-α.核糖体伴侣 NACA 招募 PHD2 进行共翻译修饰 HIF-α。
EMBO J. 2022 Nov 17;41(22):e112059. doi: 10.15252/embj.2022112059. Epub 2022 Oct 11.
3
Treatment of Alzheimer's disease with framework nucleic acids.用框架核酸治疗阿尔茨海默病。
Cell Prolif. 2020 Apr;53(4):e12787. doi: 10.1111/cpr.12787. Epub 2020 Mar 12.

本文引用的文献

1
Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translation regulation of mRNAs encoding ribosomal proteins.靶向 RNA 翻译谱分析揭示了 mTOR-4EBP1/2 非依赖性核糖体蛋白编码 mRNA 的翻译调控。
Proc Natl Acad Sci U S A. 2018 Oct 2;115(40):E9325-E9332. doi: 10.1073/pnas.1805782115. Epub 2018 Sep 17.
2
mTOR Signaling in Growth, Metabolism, and Disease.生长、代谢及疾病中的mTOR信号传导
Cell. 2017 Apr 6;169(2):361-371. doi: 10.1016/j.cell.2017.03.035.
3
The integrated stress response.整合应激反应
EMBO Rep. 2016 Oct;17(10):1374-1395. doi: 10.15252/embr.201642195. Epub 2016 Sep 14.
4
mTOR coordinates protein synthesis, mitochondrial activity and proliferation.mTOR协调蛋白质合成、线粒体活性和增殖。
Cell Cycle. 2015;14(4):473-80. doi: 10.4161/15384101.2014.991572.
5
Quantitative profiling of initiating ribosomes in vivo.体内起始核糖体的定量分析。
Nat Methods. 2015 Feb;12(2):147-53. doi: 10.1038/nmeth.3208. Epub 2014 Dec 8.
6
Translation inhibitors cause abnormalities in ribosome profiling experiments.翻译抑制剂会在核糖体分析实验中导致异常。
Nucleic Acids Res. 2014;42(17):e134. doi: 10.1093/nar/gku671. Epub 2014 Jul 23.
7
Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution.在单核苷酸分辨率下绘制哺乳动物细胞翻译起始位点的全球图谱。
Proc Natl Acad Sci U S A. 2012 Sep 11;109(37):E2424-32. doi: 10.1073/pnas.1207846109. Epub 2012 Aug 27.
8
The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments.核糖体图谱分析策略通过深度测序核糖体保护的 mRNA 片段来监测体内翻译。
Nat Protoc. 2012 Jul 26;7(8):1534-50. doi: 10.1038/nprot.2012.086.
9
Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes.鼠胚胎干细胞的核糖体图谱分析揭示了哺乳动物蛋白质组的复杂性和动态性。
Cell. 2011 Nov 11;147(4):789-802. doi: 10.1016/j.cell.2011.10.002. Epub 2011 Nov 3.
10
High-throughput real-time quantitative reverse transcription PCR.高通量实时定量逆转录PCR
Curr Protoc Mol Biol. 2006 Feb;Chapter 15:Unit 15.8. doi: 10.1002/0471142727.mb1508s73.

文献检索

告别复杂PubMed语法，用中文像聊天一样搜索，搜遍4000万医学文献。AI智能推荐，让科研检索更轻松。

立即免费搜索

文件翻译

保留排版，准确专业，支持PDF/Word/PPT等文件格式，支持 12+语言互译。

免费翻译文档

深度研究

AI帮你快速写综述，25分钟生成高质量综述，智能提取关键信息，辅助科研写作。

立即免费体验