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.
Published: May 7, 2021
Citation
Hu D., Y. Cui, L. Markillie, W.B. Chrisler, Q. Wang, R.S. Hatzenpichler, and G. Orr. 2021.Counting mRNA copies in intact bacterial cells by fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH). In Fluorescence In-Situ Hybridization (FISH) for Microbial Cells . Methods in Molecular Biology, edited by N.F. Azevedo and C. Almeida. 237-247. New York, New York:Humana.PNNL-SA-145041.doi:10.1007/978-1-0716-1115-9_15