Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide

Abstract

Understanding the composition, regulation, and function of complex biological systems requires tools that quantify multiple transcripts at their native cellular locations. However, the current multiplexed RNA imaging technologies are limited by their relatively low sensitivity or specificity, which hinders their applications in studying highly autofluorescent tissues, such as formalin-fixed paraffin-embedded (FFPE) tissues. To address this issue, here we develop a multiplexed in situ RNA profiling approach with a high sensitivity and specificity. In this approach, transcripts are first hybridized by target-specific oligonucleotide probes in pairs. Only when these two independent probes hybridize to the target in tandem will the subsequent signal amplification by oligonucleotide hybridization occur. Afterwards, horseradish peroxidase (HRP) is applied to further amplify the signal and stain the target with cleavable fluorescent tyramide (CFT). After imaging, the fluorophores are chemically cleaved and the hybridized probes are stripped by DNase and formamide. Through cycles of RNA staining, fluorescence imaging, signal cleavage, and probe stripping, many different RNA species can be profiled at the optical resolution. In applying this approach, we demonstrated that multiplexed in situ RNA analysis can be successfully achieved in both fixed, frozen, and FFPE tissues.

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

“…This in situ protein analysis method can also be combined with nucleic acids [24][25][26][27][28][29][30][31][32] and metabolic imaging technologies [33] to enable the integrated DNA, RNA, protein and metabolic profiling of single cells in intact tissues. Moreover, a program-controlled microfluidic system [34] together with a standard fluorescence microscope can be easily made into an automatic tissue imaging platform.…”

Spatially Resolved and Highly Complexed Protein and RNA in situ Detection by Combining CODEX with RNAscope In Situ Hybridization

“…Using the hotplate antigen retrieval method is easier to eliminate RNase contamination by baking glass-beaker and coverslip holder. The Comb-CODEX-RNAscope described in this paper can be further enhanced with multiplex RNAscope ISH published recently (35, 36) to maximize the detection of interested RNA signals in addition to highly multiplexed proteins.…”

Regulatory guidelines and preclinical tools to study the biodistribution of RNA therapeutics

“…Importantly, quantification of FISH data requires software that must be validated. Finally, it should be noted that that autofluorescence or aberrant probe-binding can cause an unwanted background signal (section 5.2) [96] , [109] , [113] , that FISH is time-consuming and requires some experience, and that the necessary fixation prevents using the sample in other downstream assays [96] , [114] .…”