Detection and imaging of RNA at the single-cell level is of utmost importance for fundamental research and clinical diagnostics. Current techniques of RNA analysis, including fluorescence in situ hybridization (FISH), are long, complex, and expensive. Here, we report a methodology of amplified FISH (AmpliFISH) that enables simpler and faster RNA imaging using small and ultrabright dye-loaded polymeric nanoparticles (NPs) functionalized with DNA. We found that the small size of NPs (below 20 nm) was essential for their access to the intracellular mRNA targets in fixed permeabilized cells. Moreover, proper selection of the polymer matrix of DNA-NPs minimized nonspecific intracellular interactions. Optimized DNA-NPs enabled sequence-specific imaging of different mRNA targets (survivin, actin, and polyA tails), using a simple 1 h staining protocol. Encapsulation of cyanine and rhodamine dyes with bulky counterions yielded green-, red-, and far-red-emitting NPs that were 2-100-fold brighter than corresponding quantum dots. These NPs enabled multiplexed detection of three mRNA targets simultaneously, showing distinctive mRNA expression profiles in three cancer cell lines. Image analysis confirmed the single-particle nature of the intracellular signal, suggesting single-molecule sensitivity of the method. AmpliFISH was found to be semiquantitative, correlating with RT-qPCR. In comparison with the commercial locked nucleic acid (LNA)-based FISH technique, AmpliFISH provides 8-200-fold stronger signal (dependent on the NP color) and requires only three steps vs ∼20 steps together with a much shorter time. Thus, combination of bright fluorescent polymeric NPs with FISH yields a fast and sensitive single-cell transcriptomic analysis method for RNA research and clinical diagnostics.
Keywords: DNA-functionalized polymeric nanoparticles; RNA imaging; fluorescence in situ hybridization; fluorescence microscopy; fluorescent nanoparticles; mRNA; single-cell analysis.