FlowFISH

FlowFISH is a technique that combines fluorescent in situ hybridization with flow cytometry to quantify RNA molecules within individual cells. It uses fluorescently labeled antisense probes that hybridize to target RNA in fixed and permeabilized cells, and the resulting fluorescence intensity is measured by flow cytometry. This allows researchers to estimate RNA copy number per cell and to assess expression heterogeneity across large cell populations. FlowFISH can be used with multiple probes and combined with surface or intracellular markers to profile RNA expression within defined cell subsets.

Methodology typically involves fixing and permeabilizing cells, incubating them with one or more fluorescent probes under conditions that promote specific hybridization, washing to remove nonspecific binding, and then analyzing the cells by flow cytometry. Signals can be quantified per cell and, with appropriate calibration or standards, converted into approximate transcript counts. The approach can be adapted to detect various RNA targets, including messenger RNA and ribosomal RNA, and can be performed in diverse cell types, including immune cells and certain microbes. Multiplexing is possible by using probes labeled with distinct fluorophores.

Applications of flowFISH include studying gene expression heterogeneity in complex tissues, characterizing cellular responses to stimuli, and profiling RNA content in clinical or experimental samples. It is particularly useful when single-cell resolution is required and when conventional bulk RNA methods do not capture cell-to-cell variability. Limitations include the need for fixed samples, potential background or nonspecific binding, dependence on high-quality probe design, and constraints related to fluorescence channel availability and instrument sensitivity.