X-FISH: Analysis of cellular RNA expression patterns using flow cytometry

• Updated FISH protocol for examination of mRNA expression using flow cytometry
• Demonstrate sensitive and specific detection in cell lines and primary cells
• Provide examples for analysis of cell activation and subset discrimination
• Show complementary use to antibody-based labeling protocols

Fluorescent in situ hybridization (FISH) is a powerful technique for the detection of RNA or DNA within cells and tissues, which provides a unique link between molecular and cell biology. This technique is broadly applicable across a range of biological systems. While FISH has been previously adapted to flow-based platforms, their use remains limited because of procedural challenges and costs associated with commercial kits. Herein we present a protocol that modifies existing techniques to sensitively and specifically detect and examine RNA expression patterns in primary cells and cell lines using flow cytometry (expression-FISH; X-FISH). As relevant examples, we show how this technique can be used to monitor changes in mRNA expression following activation, how it can be combined with antibody staining to study RNA and protein in the same sample, and how it can help distinguish among subsets in a mixed cell population. X-FISH can integrate multiple probes and can be performed in conjunction with other assays, allowing for informative multiparametric analyses and increased statistical robustness. For non-classical comparative animal models this procedure provides a time saving alternative to de novo production of antibody-based markers. Finally, X-FISH provides an economical solution that is applicable to conventional as well as multi-spectral imaging flow cytometry platforms.