Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution

In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization over large volumes has been largely restricted to labeling of proteins. But volumetric nucleic acid labeling may represent a far greater scientific and clinical opportunity, enabling detection of not only diverse coding RNA variants but also non-coding RNAs. Moreover, scaling immunohistochemical detection to large tissue volumes has limitations due to high cost, limited renewability/availability, and restricted multiplexing capability of antibody labels. With the goal of versatile, high-content, and scalable molecular phenotyping of intact tissues, we developed a method using carbodiimide-based chemistry to stably retain RNAs in clarified tissue, coupled with amplification tools for multiplexed detection. The resulting technology enables robust measurement of activity-dependent transcriptional signatures, cell-identity markers, and diverse non-coding RNAs in rodent and human tissue volumes. The growing set of validated probes is deposited in an online resource for nucleating related developments from across the scientific community. [Display omitted] •Carbodiimide-based chemistry in CLARITY hydrogels for RNA preservation and detection•Rapid diffusion of DNA oligonucleotides for volumetric in situ hybridization•Detection of microRNAs and mRNAs in clarified mouse and human tissues•DNA-based amplification for multiplexed in situ hybridization in CLARITY CLARITY with enhanced RNA coupling chemistry is developed for multiplexed, volumetric visualization of both long and short RNAs in a variety of intact tissues.