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.
CITATION STYLE
Hu, D., Cui, Y., Markillie, L. M., Chrisler, W. B., Wang, Q., Hatzenpichler, R., & Orr, G. (2021). Counting mRNA Copies in Intact Bacterial Cells by Fluctuation Localization Imaging-Based Fluorescence In Situ Hybridization (fliFISH). In Methods in Molecular Biology (Vol. 2246, pp. 237–247). Humana Press Inc. https://doi.org/10.1007/978-1-0716-1115-9_15
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