In Situ Sequencing: A High-Throughput, Multi-Targeted Gene Expression Profiling Technique for Cell Typing in Tissue...

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Image-based in situ mRNA quantification methods visualize gene expression spatially within tissues.
• Existing techniques enable parallel mapping of numerous genes and assignment to specific cells by capturing cellular boundaries.

Purpose of the Study:

• To present a high-throughput, multi-targeted gene expression profiling technique named in situ sequencing.
• To demonstrate the capability of in situ sequencing for localizing hundreds of genes simultaneously.
• To support cell type classification based on transcriptome-based taxonomy.

Main Methods:

• In situ sequencing utilizes padlock probes (PLPs) and rolling circle amplification (RCA) for targeted, amplified, and barcoded gene detection.
• The protocol involves mRNA fixation, reverse transcription, degradation of residual mRNA, and PLP hybridization.
• Amplified PLPs are labeled with fluorophore-conjugated probes for detection using conventional fluorescence microscopes.

Main Results:

• Successfully localized hundreds of genes simultaneously within tissue sections.
• Enabled cell type classification aligned with transcriptome-based taxonomy.
• Demonstrated the utility of in situ sequencing for high-resolution spatial gene expression analysis.

Conclusions:

• In situ sequencing is a powerful method for high-throughput, multi-targeted gene expression profiling.
• The technique provides spatial resolution for gene localization and facilitates cell type identification.
• This approach advances the understanding of gene expression patterns in complex biological systems.