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Single-molecule analysis of gene expression using two-color RNA labeling in live yeast


Live-cell imaging of mRNA yields important insights into gene expression, but it has generally been limited to the labeling of one RNA species and has never been used to count single mRNAs over time in yeast. We demonstrate a two-color imaging system with single-molecule resolution using MS2 and PP7 RNA labeling. We use this methodology to measure intrinsic noise in mRNA levels and RNA polymerase II kinetics at a single gene.

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Figure 1: Expression of MDN1 alleles fluctuate independently over time.
Figure 2: Two-color transcriptional induction as a readout for polymerase dynamics.

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We thank H.Y. Park, S.J. Gandhi, T. Lionnet and D.R. Larson for helpful discussions and feedback. This work was supported by the US National Institutes of Health (GM57071 to R.H.S. and National Research Service Award individual fellowship F32GM083430 to J.A.C.), Canadian Institutes of Health Research (MOP-BMB-232642 to D.Z.), Fonds de recherche du Québec–Santé (Chercheur Boursier to D.Z.) and funding from the Canada Foundation for Innovation.

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Experiments and data analysis were performed by S.H. and P.R. Tagging constructs and coat protein expression vectors were created by D.Z., J.A.C., S.H. and P.R. The manuscript was written by S.H. Project consultation and guidance were provided by D.Z., J.A.C. and R.H.S.

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Correspondence to Robert H Singer.

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The authors declare no competing financial interests.

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Hocine, S., Raymond, P., Zenklusen, D. et al. Single-molecule analysis of gene expression using two-color RNA labeling in live yeast. Nat Methods 10, 119–121 (2013).

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