通过多核糖体谱分析测量mRNA翻译

Measuring mRNA Translation by Polysome Profiling.

作者信息

Kudla Marek, Karginov Fedor V

机构信息

Department of Cell Biology and Neuroscience, University of California, Riverside, 900 University Avenue, Riverside, CA, 92521, USA.

出版信息

Methods Mol Biol. 2016;1421:127-35. doi: 10.1007/978-1-4939-3591-8_11.

DOI:10.1007/978-1-4939-3591-8_11

PMID:26965262

Abstract

Determination of mRNA translation rates is essential to understanding the regulatory pathways governing eukaryotic gene expression. In this chapter, we present a transcriptome-wide method to assess translation by association of mRNAs with polysomes on sucrose density gradients. After sedimentation, the fractions are spiked with a control RNA mixture and the RNA content is measured by high-throughput sequencing. Normalization to the spike-ins provides a global quantitative view on the translational status of cellular mRNAs, with the ability to measure changes and identify active and silent subpopulations of each.

摘要

确定mRNA翻译速率对于理解真核基因表达的调控途径至关重要。在本章中，我们介绍了一种全转录组范围的方法，通过蔗糖密度梯度上mRNA与多核糖体的结合来评估翻译。沉降后，向各组分中加入对照RNA混合物，并通过高通量测序测量RNA含量。与内参RNA的标准化提供了细胞mRNA翻译状态的全局定量视图，能够测量变化并识别每个mRNA的活跃和沉默亚群。