Fig. 1: RNAs can be reproducibly and accurately detected in myofibers by HCR FISH and are dispersed in the myofiber cytoplasm.

A Schematic describing experimental strategy to label RNAs and proteins of interest in adult skeletal muscle. B Transcript length (nucleotides, nt) and abundance in tibialis anterior muscle (transcripts per million, TPM) for each RNA studied; colors represent encoded protein localization. C Representative FISH images for each RNA studied. Scale bars: 5 μm. D RNA density (spots/µm³) measured from FISH images compared with transcripts per million (TPM) values from a tibialis anterior RNAseq dataset. Dotted line is lower limit of detection (LLOD). Trendline: LLS regression; Pearson R = 0.98; p < 0.05, Wald test. E RNA densities compared across separate experiments. Black bars are the mean ± s.d. of RNA density. Experiment 1: n = 10 myofibers (Polr2a, Hist1h1c, Ttn, GFP) or n = 9 myofibers (Vcl, Dmd, Hnrnpa2b1, Myom1, Gapdh). Experiment 2: n = 3 myofibers. Trendline: LLS regression; Pearson R = 0.97; p < 0.05, Wald test. F Schematic describing percent dispersion calculation with example cumulative distribution function (CDF) for Polr2a RNA. G Percent dispersion for each RNA studied, points are colored as in Fig. 1B. Dotted line indicates fully uniform dispersion. H Mean Gapdh FISH signal intensity of myofiber in C plotted for 40 µm along the longitudinal axis (top). The power spectral density of this signal (bottom).