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US 2026/0308668 A1Published application
Methods and Systems for Sensitive and Multiplexed Analysis of Short RNA Sequences
AssigneeArizona Board of Regents on behalf of Arizona State University
InventorJia GUO
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Abstract
Methods for multiplexed single-cell spatial nucleic acid profiling in intact cells are described herein. The methods enhance multiplexed nucleic acid staining specificity and imaging sensitivity by several orders of magnitude. Also provided are novel probes and compounds for use in the methods.
Claims19
A method for multiplexed in situ analysis of nucleic acids in a tissue, the method comprising:
- a) contacting the tissue with a target probe or a pair of target probes capable of hybridizing to a target nucleic acid;
- b) contacting the tissue with a signal amplification molecule capable of binding to the target probe or pair of target probes, wherein the signal amplification molecule comprises a plurality of horseradish peroxidase (HRP) molecules;
- c) contacting the tissue with a cleavable hapten tyramide (CHT) in a tyramide signal amplification (TSA) buffer;
- d) contacting the tissue with HRP-conjugated streptavidin (SA-HRP);
- e) contacting the tissue with a cleavable fluorophore tyramide (CFT) in a TSA buffer;
- f) imaging the tissue;
- g) contacting the tissue with an HRP deactivating agent;
- h) contacting the tissue with biotin;
- and
- i) repeating steps a) through h) for a plurality of rounds, wherein in each round, the target probe is capable of hybridizing to a different target nucleic acid;
- and wherein the fluorophore of each CFT is different than an immediately previous round.
8 more elements
A cleavable hapten tyramide (CHT) comprising formula I)
- wherein R1 is
Dependencies are inferred from the claim text.