PrimeFlow^TM Assay for Cell Type-Specific Co-detection of Transgene RNA and Protein in Mouse Spleens From Preclinical...

The PrimeFlow^TM assay is a flow cytometry-based method for the co-detection of RNAs and proteins in cells. When combined with cell characterization by immunophenotyping, PrimeFlow^TM can be used to simultaneously detect RNA and proteins in a cell type-specific manner in complex heterogeneous samples, offering an advantage over bulk tissue analysis methods. Here, we describe the implementation of the PrimeFlow^TM assay protocol for the detection of transgene mRNA and protein expression in spleen samples from mice treated in vivo with luciferase mRNA-lipid nanoparticles (LNPs). This protocol involves spleen tissue dissociation for cell isolation, followed by cell fixation and permeabilization to allow immunolabeling of intracellular luciferase protein. The immunophenotyping strategy is based on immunolabeling with mouse CD marker antibodies for the identification of T cells, B cells, monocytes, granulocytes/macrophages, NK cells, and non-hematopoietic cells. The RNAs of luciferase and a housekeeping gene, β-actin, are detected with sequence-specific probe sets by employing sequential oligonucleotide annealing steps and fluorescent labeling using a branched DNA (bDNA) technology. Samples are analyzed by flow cytometry. Based on our analysis, we conclude it is feasible to apply the PrimeFlow^TM approach for evaluating successful drug targeting to the cell types of interest and any potential differences in the kinetics of RNA delivery and protein expression in various tissue cells, supporting the discovery and development of RNA therapeutics. Key features • This protocol can be employed for cell-specific simultaneous detection of up to four different RNAs (using available commercial reagents) and multiple proteins by flow cytometry. • This protocol requires application-specific optimization of the RNA-binding probe sets and antibody reagents for transgene detection and immunophenotyping panel.