期刊
NATURE PROTOCOLS
卷 8, 期 6, 页码 1100-1113出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2013.066
关键词
-
资金
- US National Institutes of Health [R01 GM082837]
- US National Science Foundation [082265, PHY-1147498]
- Human Frontier Science Program [RGY 70/2008]
- Welch Foundation [Q-1759]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM082837] Funding Source: NIH RePORTER
- Direct For Mathematical & Physical Scien [1147498] Funding Source: National Science Foundation
We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The copy number of the target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete 'spots' as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 d, with another 2-3 d typically required for image and data analysis.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据