Fig. 2

Benchmarking of a combined PEA/STA workflow: AXIN1 and MKI67. a Two-fold dilutions of bulk population lysate (top) and recombinant AXIN1 protein (bottom) were backloaded into the C1 IFC and detected using the same reactions conditions employed in the PEA/STA protocol. Each data point plotted is the average of eight replicates and error bars show the standard error of the mean. Points used for fitting the red trend line are colored blue. Gray (green) dashes show the level above which the probability for a detection event being real is p = 0.01 (0.05). b–d Validation of protein and RNA detection in single cells using a coupled PEA/STA script on the C1 throughout a PMA perturbation time course (0 hr = purple, 24 hr = green, 48 hr = blue). b RNA fluorescence in situ hybridization (RNA-FISH) and protein IF staining of MKI67 RNA and protein was performed to validate the C1-based, high-throughput RNA and protein measurements. Cyan (left) shows cell nuclei and boundaries, magenta MKI67 protein (middle), and yellow MKI67 RNA (right). Scale bars indicate 25 μm. c Qualitative agreement between the protein and RNA data obtained in situ and on the C1. Density distributions (each with their own arbitrary units) for MKI67 RNA (left) and protein (right) obtained via qPCR (top) or in situ (bottom) staining. d Quantile-Quantile (Q-Q) plots showing the range over which the PEA/STA measurements of MKI67 protein and RNA track linearly with IF staining or in situ hybridization