Dual probe ligation in situ hybridization with rolling-circle amplification for high-plex spatial transcriptomics.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Understanding tissue architecture is crucial for biological insights.
• Spatial transcriptomics technologies are vital for studying tissue architecture.
• Formalin-fixed and paraffin-embedded (FFPE) tissues are widely available but challenging for transcriptomics due to mRNA degradation.

Purpose of the Study:

• To develop a streamlined method for spatial transcriptomics analysis of FFPE tissues.
• To accurately detect specific mRNA isoforms within FFPE tissue architectures.
• To enable highly multiplexed spatial transcriptomic studies on clinical specimens.

Main Methods:

• Ligation In Situ Hybridization followed by rolling circle amplification (LISH-LnR) was developed.
• Iterative fluorescent probe hybridization and imaging were employed for multiplexed detection.
• Molecular rheostats were used to fine-tune assay performance.

Main Results:

• LISH-LnR accurately detects spatial locations of specific mRNA isoforms in FFPE tissues.
• The method was successfully demonstrated on FFPE specimens from inclusion body myositis and pediatric rhabdomyosarcoma patients.
• The LISH-LnR assay performance was successfully fine-tuned using molecular rheostats.

Conclusions:

• LISH-LnR provides a powerful toolkit for spatial transcriptomics on FFPE clinical specimens.
• This methodology enhances the analysis of degraded mRNA in challenging tissue types.
• Combined with LISH-seq and LISH-QC, LISH-LnR advances the field of spatial transcriptomics.