Fig. 2: WM-smFISH enables combining of RNA and protein quantification.

a, Schematic diagram for simultaneous RNA and protein detection. VENUS mRNAs are hybridized and detected with smFISH probes and the VENUS proteins are detected directly through protein fluorescence. b, Transition embryo expressing pDR5rev::3xVENUS-N7 showing detection of VENUS mRNA (magenta) and VENUS protein (green). c, Close-up of a single cell from the embryo presented in b, showing individual mRNAs as single spots and VENUS protein fluorescence in the nucleus. d, Workflow diagram showing the three-stepped pipeline for quantitative analysis of WM-smFISH with fluorescent protein detection. e–h, Simultaneous mRNA and protein detection in heart stage embryo (e) and leaf (g) using pDR5rev::3xVENUS-N7 and pCUC2::3xVENUS-N7 reporter lines, respectively. Confocal microscopy images for mRNA (magenta), protein (green), and merged signals. Quantification results for mRNA and protein in heart stage embryo (f) and leaf (h). (i, ii) Heatmaps represent the levels of the mean signal intensity per cell detected in each channel (for RNA or protein detection). (iii) Heatmap representing the ratio between the RNA and protein signal intensities per cell. (iv) Histograms showing the distribution of the number of transcripts (magenta) or total protein intensity (green) per cell, the median value is indicated with a dashed line. The contours of cells were visualized with Renaissance 2200 dye (white). Scale bars, 20μm. Experiments were repeated independently at least three times.