Sensitive Spatially Multiplexed Profiling of miRNA in Plant Tissue Using Multispectral Rolling Circle Amplification in Nanoliter Well Arrays.

Quantifying the spatial distribution of microRNAs (miRNAs) within plant tissue is critical for deciphering cell-specific gene-regulatory pathways. While numerous miRNAs are involved in plant response pathways, traditional spatial miRNA detection techniques are limited to simultaneously profiling two miRNAs with a limited dynamic range. We introduce an advanced spatial profiling approach using multispectral imaging in which miRNAs are uniquely identified with spectrally distinct, minimally overlapping fluorophores. Spectral multiplexing is performed using functionalized DNA probes fixed within hydrogel posts located in nanoliter well arrays that contain both miRNA-specific and fluorophore-specific regions, enabling concurrent labeling using five fluorophores. We integrate spectral multiplexing with rolling circle amplification (RCA) to improve sensitivity and dynamic range by amplifying binding events using a DNA concatemer specific to each fluorophore. We achieved multiplex miRNA detection using five unique fluorophores, with a fluorophore-specific detection limit ranging from 1.9 to 19 zeptomoles, all within a single gel post. We showed that multispectral RCA enhances detection and quantifies low-abundance targets in ethanol-fixed, paraffin-embedded Arabidopsis thaliana leaf sections. Integrating multispectral RCA with our nanoliter well array enhances multiplexing by 5-fold per post and expands the utility of the technique for miRNA spatial profiling in plant tissue.