Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization.

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Understanding transcript and genomic loci copy numbers in spatial contexts is crucial for biology and medicine.
• Fluorescent in situ hybridization is a powerful tool for single-cell analysis but is limited in the number of quantifiable nucleic acid species.
• Current methods face limitations in simultaneously profiling a large number of different nucleic acid targets within their cellular context.

Purpose of the Study:

• To develop a highly multiplexed in situ hybridization approach for spatial transcriptomics and genomics analysis.
• To overcome the limitations of existing fluorescence imaging-based methods in quantifying multiple nucleic acid species.
• To enable precise profiling of tens of thousands of different transcripts or genomic loci in individual cells in situ.

Main Methods:

• Developed a reiterative fluorescence in situ hybridization technique.
• Each nucleic acid molecule is visualized as a fluorescent spot in its natural cellular context.
• Utilized consecutive cycles of hybridization, imaging, and photobleaching to generate unique color sequence barcodes for varied nucleic acids.

Main Results:

• Demonstrated a highly multiplexed approach for spatial transcriptomics and genomics.
• Successfully visualized and quantified numerous nucleic acid targets within individual cells.
• Achieved precise profiling of tens of thousands of different transcripts or genomic loci through multi-color staining and reiterative cycles.

Conclusions:

• The developed method significantly expands the capability of in situ nucleic acid quantification.
• This approach allows for unprecedented resolution in spatial transcriptomics and genomics within single cells.
• Offers a powerful new tool for advancing biological and medical research through detailed spatial profiling.