Figure 5. Stochastic profiling distinguishes heterogeneous expression patterns that are not exclusively coexpressed.

Two-color RNA FISH images were collected at day 10 of MCF10A morphogenesis and compared to the stochastic-profiling data for (a-c) FOXO1 and IL1R1, (d-f) IL1R1 and JUND, and (g-i) FOXO1 and JUND. (a,d,g) Representative pseudocolored images, containing single-positive cells highlighted with red arrows (high expression) or flat markers (low expression) Scale bar is 20 μm. (b,e,h) Fluorescence intensities are shown from cells in four independent acini after normalization to the maximum observed cellular fluorescence signal in each image. Gates were defined as the 25^th percentile centered on the median fluorescence intensity (black lines) for each gene. Observations that were within the range of the gates were scored as neither positive nor negative (gray). The percentages of single-positive cells (red) in the overall cell population are shown with 90% confidence intervals in parentheses. (c,f,i,) RNA FISH distributions of b,e,h were resampled as 10-cell averages, standardized, and the resulting probability density function (pdf) was compared to the standardized sampling fluctuations from stochastic profiling (black circles) shown in Figure 3b.