title: Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution.
abstract: 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.
authors.affiliation: Department of Bioengineering, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA.
authors.forename: Emily Lauren
authors.initials: EL
authors.lastname: Sylwestrak
authors.affiliation: Department of Bioengineering, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA.
authors.forename: Priyamvada
authors.initials: P
authors.lastname: Rajasethupathy
authors.affiliation: Department of Bioengineering, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA; Department of Psychiatry and Behavioral Sciences, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA.
authors.forename: Matthew Arnot
authors.initials: MA
authors.lastname: Wright
authors.affiliation: Department of Bioengineering, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Jaffe
authors.affiliation: Department of Bioengineering, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA; Department of Psychiatry and Behavioral Sciences, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA. Electronic address: deissero@stanford.edu.
authors.forename: Karl
authors.initials: K
authors.lastname: Deisseroth
mesh_terms: D000293:Adolescent; D000818:Animals; D001923:Brain Chemistry; D003485:Cyanates / Q000737:chemistry; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D005260:Female; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D008301:Maleimides / Q000737:chemistry; D051379:Mice; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry; D012313:RNA / Q000032:analysis*; D013388:Succinimides / Q000737:chemistry; D059467:Transcriptome
chemical_list: D003485:Cyanates; D008301:Maleimides; D009841:Oligonucleotides; D013388:Succinimides; C082056:4-maleimidophenyl isocyanate; D012313:RNA; D005022:Ethyldimethylaminopropyl Carbodiimide; C019358:disuccinimidyl suberate