Subcellular Imaging of the Entire Protein Coding Human Transcriptome on FFPE Tissue Using Spatial Molecular Imaging

For the very first time, subcellular imaging of the entire human protein coding transcriptome has been accomplished. Using a fluorescence in situ hybridization approach, we developed 37,968 imaging probes to detect 18,934 mRNA transcripts without use of enzyme-amplification and achieve subcellular resolution (38 nm +/- 11 nm) of transcripts. Data is presented for formalin-fixed paraffin-embedded (FFPE) tissues (hippocampus region of brain, skin, colon, breast, pancreas, kidney) that average (across all tissues) over 2,000 transcripts per cell and over 1,100 unique genes per cell (highest transcript number/diversity ever imaged). A total of 5.5 billion transcripts measured in 2.7 million single cells. From one 5 µm thick FFPE section, this method can measure all subcellular RNA localizations, explore every protein-based biological pathway, every ligand-receptor interaction, analyze pseudotime RNA trajectories, and predict spatial copy number variations. Unbiased spatial molecular discovery on archival human tissue empowers biological discovery via reverse translation, and new capabilities for drug development, companion diagnostics, and therapeutic intervention. Whole transcriptome sub-cellular imaging establishes a new genomic standard for single cell and spatial analysis.