Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.
Nat Protoc. 2013 Jun;8(6):1100-13. doi: 10.1038/nprot.2013.066. Epub 2013 May 16.
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.
我们提出了一种在单个化学固定的大肠杆菌细胞中测量目的基因的 mRNA 分子绝对数量的方案。一组荧光标记的寡核苷酸探针与靶 mRNA 杂交，使得每个 mRNA 分子被已知数量的荧光染料修饰。然后使用荧光显微镜对细胞进行成像。目标 mRNA 的拷贝数是从细胞中荧光焦点的总强度估计的，而不是像其他当前可用的方案那样通过计数离散的“斑点”来估计。图像分析使用自动算法进行。从许多单个细胞中获得的测量的 mRNA 拷贝数分布可用于提取随机基因活性的参数，即来自感兴趣基因的转录爆发的频率和大小。实验过程需要 2 天，另外通常还需要 2-3 天用于图像和数据分析。
