The circadian oscillator analysed at the single‐transcript level

The circadian clock is an endogenous and self‐sustained oscillator that anticipates daily environmental cycles. While rhythmic gene expression of circadian genes is well‐described in populations of cells, the single‐cell mRNA dynamics of multiple core clock genes remain largely unknown. Here we use single‐molecule fluorescence in situ hybridisation (smFISH) at multiple time points to measure pairs of core clock transcripts, Rev‐erbα (Nr1d1), Cry1 and Bmal1, in mouse fibroblasts. The mean mRNA level oscillates over 24 h for all three genes, but mRNA numbers show considerable spread between cells. We develop a probabilistic model for multivariate mRNA counts using mixtures of negative binomials, which accounts for transcriptional bursting, circadian time and cell‐to‐cell heterogeneity, notably in cell size. Decomposing the mRNA variability into distinct noise sources shows that clock time contributes a small fraction of the total variability in mRNA number between cells. Thus, our results highlight the intrinsic biological challenges in estimating circadian phase from single‐cell mRNA counts and suggest that circadian phase in single cells is encoded post‐transcriptionally.