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

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Understanding biological systems necessitates tools for quantifying multiple transcripts in their native cellular locations.
• Current multiplexed RNA imaging technologies face limitations in sensitivity and specificity, particularly in autofluorescent tissues like FFPE.

Purpose of the Study:

• To develop a highly sensitive and specific multiplexed in situ RNA profiling approach.
• To overcome limitations of existing methods for analyzing RNA in challenging tissue types.

Main Methods:

• Utilizes target-specific oligonucleotide probes in pairs for initial hybridization.
• Employs signal amplification via oligonucleotide hybridization and horseradish peroxidase (HRP) with cleavable fluorescent tyramide (CFT).
• Incorporates cyclic RNA staining, imaging, signal cleavage, and probe stripping for multiplexed analysis.

Main Results:

• Demonstrates high sensitivity and specificity in multiplexed in situ RNA profiling.
• Successfully applied the approach to fixed, frozen, and formalin-fixed paraffin-embedded (FFPE) tissues.
• Enables profiling of diverse RNA species at optical resolution.

Conclusions:

• The developed method offers a significant advancement for in situ RNA analysis.
• It is effective across various tissue preservation states, including FFPE.
• This technique expands the possibilities for studying complex biological systems at the transcriptomic level.