Quantitative and spatially resolved detection of multiplexed microRNA from plant tissue via hybridization to hydrogel-bound DNA probes in nanoliter well arrays.

Understanding complex regulatory networks in plant systems requires elucidating the roles of various gene regulators under a spatial landscape. MicroRNA are key regulators that impart high information value through their tissue specificity and stability when using expression patterns for evaluating network outcomes. However, current techniques that utilize spatial multiplexing and quantitation of microRNA are limited to primarily mammalian systems. Here, we present a method to spatially resolve and quantify multiple endogenous microRNA in situ using ethanol fixed, paraffin embedded model plant species. This method utilizes target-specific microRNA capture along with universal ligating and labelling, all within functionalized hydrogel posts containing DNA probes in nanoliter well arrays. We demonstrate the platform's multiplexing capabilities through analyzing three endogenous microRNA in Arabidopsis thaliana rosettes which provide useful answers to fundamental plant growth and development from the unique expression patterns. The spatial tissue technique is also validated using non-spatial small RNA assays to demonstrate the versatility of the well array platform. Our new platform expands the toolkit of spatial omics technologies for plants.