Mapping Human Tissues with Highly Multiplexed RNA in situ Hybridization

Area of Science:

• Molecular Biology
• Genomics
• Neuroscience

Background:

• Multiplexed RNA in situ hybridization (ISH) techniques offer insights into tissue organization.
• Existing multiplexed ISH methods face limitations in human biopsies due to tissue size, quality, and autofluorescence.

Approach:

• Developed DART-FISH, a padlock probe-based technology for profiling hundreds to thousands of genes in large human tissue sections at cellular resolution.
• Introduced RiboSoma, a cytoplasmic stain for improved cell body segmentation.
• Implemented a computational decoding-by-deconvolution workflow to resolve optical crowding.
• Utilized an enzyme-free isothermal decoding procedure for rapid imaging.

Key Points:

• Successfully imaged 121 genes in a human neocortex section in under 10 hours, recapitulating cytoarchitecture.
• Detected transcripts as short as 461 nucleotides, including neuropeptides, and identified novel cortical layer markers.
• Mapped 300 genes in a diseased human kidney, profiling over 20 cell states and identifying disease-associated niches.

Conclusions:

• DART-FISH is a versatile technology for transcriptomic profiling of large human tissues.
• The method enhances cellular resolution and overcomes common challenges in clinical biopsy analysis.
• Enables discovery of novel biomarkers and characterization of cellular states in health and disease.