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Updated: Jun 16, 2025
Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection
Published on: July 6, 2022
Sequencing-free whole genome spatial transcriptomics at molecular resolution in intact tissue.
Yubao Cheng1, Shengyuan Dang1,2, Yuan Zhang1,2
1Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA.
Researchers developed Reverse-padlock Amplicon Encoding FISH (RAEFISH), a new spatial transcriptomics technology. RAEFISH achieves whole-genome coverage and single-molecule resolution, enabling comprehensive spatial profiling of RNA in intact tissues.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Spatial transcriptomics technologies have advanced understanding of cellular functions but face limitations in transcriptomic coverage or spatial resolution.
- Current methods offer either broad coverage with lower resolution (in vitro sequencing) or high resolution with limited coverage (in situ hybridization/sequencing).
- These limitations hinder unbiased, high-resolution transcriptomic analyses in intact tissues.
Purpose of the Study:
- To develop a novel image-based spatial transcriptomics technology with both whole-genome coverage and single-molecule resolution.
- To enable comprehensive spatial profiling of RNA species in intact tissues for unbiased biological discovery.
- To demonstrate the technology's versatility, including its application in CRISPR screening.
Main Methods:
- Developed Reverse-padlock Amplicon Encoding FISH (RAEFISH), an image-based spatial transcriptomics technique.
- RAEFISH utilizes padlock probes and amplicon encoding for high-throughput RNA detection.
- Applied RAEFISH to profile thousands of transcript species in human and mouse tissues and cell cultures.
Main Results:
- Achieved whole-genome level transcriptomic coverage with single-molecule spatial resolution in intact tissues.
- Successfully mapped over 23,000 human and 22,000 mouse transcript species, including protein-coding and long non-coding RNAs.
- Revealed differential subcellular transcript localization, cell-type-specific transcriptome variations, and gene expression patterns related to cell-cell interactions.
Conclusions:
- RAEFISH overcomes existing limitations in spatial transcriptomics, providing unprecedented coverage and resolution.
- The technology enables detailed spatial analysis of the transcriptome in various biological contexts.
- RAEFISH is a broadly applicable tool for RNA profiling and has been extended for CRISPR screening applications.
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