Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA.
MIT Media Lab, Cambridge, MA, USA.
Methods Mol Biol. 2021;2246:237-247. doi: 10.1007/978-1-0716-1115-9_15.
A method for measuring mRNA copies in intact bacterial cells by fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) is presented. Unlike conventional single-molecule FISH, where the presence of a transcript is determined by fluorescence intensity, fliFISH relies on On-Off duty cycles of photo-switching dyes to set a predetermined threshold for distinguishing true signals from background noise. The method provides a quantitative approach for detecting and counting true mRNA copies and rejecting false signals with high accuracy.
本文提出了一种通过基于波动定位成像的荧光原位杂交（fliFISH）测量完整细菌细胞中 mRNA 拷贝数的方法。与传统的单分子 FISH 不同，传统的单分子 FISH 通过荧光强度来确定转录本的存在，而 fliFISH 依赖于光开关染料的 On-Off 工作循环来设定区分真实信号和背景噪声的预定阈值。该方法提供了一种定量检测和计数真实 mRNA 拷贝数并准确剔除假信号的方法。
