Supplementary Figure 9: DSP RNA detection LOD estimation based on RNAscope.
From: Multiplex digital spatial profiling of proteins and RNA in fixed tissue
a, Calculations performed to estimate the LOD of the DSP RNA assay. The average number of cells (nuclei counted) in the 4 replicate ROIs analyzed in Suppl. Fig. 8 are shown on the right. These cell counts were multiplied by the total number of RNAscope spots (transcripts) counted to estimate the number of transcripts present in the ROIs profiled with DSP. Three targets detected just above background are highlighted in yellow. The average of these RNA transcripts predicted to be present in these ROIs was 606 transcripts (or ~2 copies per cell across a total of ~300 cells). b, Because our LOD appeared to be around 600 transcripts, we estimate that DSP should be able to detect ~30 transcripts per cell across ~20 cells. To test this, we profiled smaller, 50 µm diameter circle ROIs. We found that expression was readily detectable down to 21 cells, thus confirming our LOD for the higher expressors examined in this study was approximately 20 cells.
