Profiling lamina specific pyramidal neurons using postmortem human formalin fixed paraffin embedded frontal cortex tissue in combination with digital spatial profiling.

BACKGROUND

Digital spatial profiling (DSP) is an innovative approach to perform RNA sequencing (RNA-seq), including in neuronal populations. DSP enables expression profiling linking RNA-seq data to spatially characterized samples utilizing tissue bound probes. We employ the GeoMx DSP system for spatial characterization of transcriptomic data from lamina specific pyramidal neurons and cortical ribbons containing admixed cell types using human postmortem brain tissue. New method We established a protocol using human postmortem formalin fixed paraffin embedded (FFPE) frontal cortex tissue from nondemented human control brains. Layer III (L3) and Layer V (L5) pyramidal neurons from Brodmann area 9 were identified with the neuronal marker Ca /calmodulin-dependent protein kinase II and selected for probe collection.

RESULTS

This approach significantly reduced the amount of FFPE tissue needed for robust single population RNA-seq. We demonstrate ∼20 identified L3 or L5 pyramidal neurons or one lamina-specific cortical ribbon from a single 5 µm thick section is sufficient to generate robust RNA-seq reads. Bioinformatic analysis of neurons and ribbons showed notable similarities and differences reflective of the single neuron and multiple admixed cell types within the former and latter, respectively. Comparison with existing methods Protocols exist for DSP of postmortem human FFPE brain tissue. However, this new approach enables profiling small groups of ∼14-21 pyramidal neurons using the GeoMx DSP platform.

CONCLUSIONS

This optimized DSP assay provides high resolution RNA-seq data demonstrating utility and versatility of the GeoMx platform for individually characterized neurons and isolated cortical ribbons within postmortem FFPE human brain tissue for downstream analyses.