GeoMx and RNAscope: A Comparative Assessment of Their Utility for Spatial mRNA Expression Profiling in Formalin-Fixed...

mRNA expression analysis in formalin-fixed tissue is essential for many biomarker and tumor microenvironment studies. NanoString GeoMx Digital Spatial Profiler is a recent technique that offers high-plex spatial transcriptomics (up to 18,000 genes), while RNAscope (1-4 plex) is a well-established mRNA in situ hybridization method. This study compares quantitative expression estimates obtained by GeoMX and RNAscope. Serial sections from two TMAs containing mammary cancers, including triple-negative (n = 48) and varying ER/HER2 status (n = 45), were analyzed using GeoMx Cancer Transcriptomics Atlas (CTA) covering approximately 1,800 genes and RNAscope probes for GATA3, SOX10, and PD-L1 mRNAs. Expression was quantified as counts/cell (GeoMx, geometric mean of five probes per gene) and average dots/cell (RNAscope, QuPath). Positivity was defined as above the limit of quantification (GeoMx) or > 0.1 dots/cell (RNAscope). High correlation was observed for GATA3 (R = 0.87) and SOX10 (R = 0.77) between methods. PD-L1 expression was high in only one core, precluding correlation analysis. RNAscope demonstrated higher sensitivity and a broader dynamic range than GeoMx. In conclusion, GeoMx CTA and RNAscope exhibit a strong correlation. GeoMx enabled highly multiplexed gene expression analysis, whereas RNAscope provided better sensitivity and single-cell resolution. The choice of method should be guided by study objectives.