Rigor and Reproducibility of GeoMx Digital Spatial mRNA Profiling performed on Human Kidney Tissues

Spatial transcriptomic profiling enables precise quantification of gene expression with simultaneous localization of expression profiles onto tissue structures. This new technology promises to improve our understanding of the disease mechanisms. Therefore, there is intense interest in applying these methods in clinical trials or as laboratory developed tests to aid in diagnosis of disease. Before these applications can been more broadly deployed in clinical research and diagnostics, it is necessary to thoroughly understand the technology’s performance in real world conditions. In this study, we vet technical reproducibility, data normalization methods and assay sensitivity of digital spatial profiling, one widely used spatial transcriptomic methodology. Using clinically sourced human tissue specimens, we find that digital spatial profiling exhibits high rigor and reproducibility. Our approach lays the foundation for incorporation of digital spatial profiling methods into clinical workflows. Human nephrectomy tissues were fixed in formalin and paraffin embedded (FFPE) and multiple tissue chunks were embedded into one block (multi-tissue block). Four consecutive 5µm thick sections from the kidney multi-tissue block were applied to charged slides and baked at 60°C for 1 hour. After deparaffinization and rehydration, sections were subjected to heat induced antigen retrieval with Tris EDTA, pH 9 for 15 minutes followed by 1µg/ml proteinase K digestion for 15 minutes at 37°C. All 4 slides were hybridized overnight at 37°C with the Cancer Transcriptome Atlas (CTA) probe mix encompassing 1,825 genes. Per Nanostring’s protocol, sections are usually incubated with 250µl of probe mix and coverslipped using a 40x22 mm RNase-free HybriSlip coverslip (ThermoFisher). However, in order to extend the use of the probes, which are the most expensive component of the digital spatial profiling workflow, we utilized 100µl of probe mix per slide and coverslipped sections using a 22x22mm HybriSlip. The following day, all 4 slides were subjected to stringency washes and counterstained with fluorescently labeled antibodies recognizing pan-cytokeratin (Cy3 channel), CD10 (Cy5 channel) as well as DNA (Syto13, FITC channel). One slide was then loaded into the instrument while the others were stored in 2x SSC in the dark at 4°C for staggered collections. On 3 subsequent days, one of the remaining labeled slides was restained with Syto13 for 10 minutes before loading onto the instrument. After loading onto the instrument and scanning, ROI selection was performed, and UV-released barcode probes were collected in staggered fashion into a 96-well collection plate (24 ROI/day). After 4 consecutive days of ROI selection, libraries were generated using a SeqCode construction kit from Nanostring. The libraries were sequenced using a NextSeq 2000 P2 flowcell (100 cycle kit). Sequenced barcodes were mapped to genes and ROIs using the Nanostring GeoMx NGS Pipeline, version 2.0.21.