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Imaging individual mRNA molecules using multiple singly labeled probes


We describe a method for imaging individual mRNA molecules in fixed cells by probing each mRNA species with 48 or more short, singly labeled oligonucleotide probes. This makes each mRNA molecule visible as a computationally identifiable fluorescent spot by fluorescence microscopy. We demonstrate simultaneous detection of three mRNA species in single cells and mRNA detection in yeast, nematodes, fruit fly wing discs, and mammalian cell lines and neurons.

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Figure 1: Simultaneous detection of a unique sequence and a repeated sequence in individual mRNA molecules.
Figure 2: Simultaneous imaging of three different mRNAs in mammalian cells.
Figure 3: Imaging localized mRNAs in C. elegans and D. melanogaster.

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We thank S.A.E. Marras, F.R. Kramer, D. Vargas, S. Sinha, E. Andersen, G. Neuert and Q. Yang. This work was supported by the US National Institute of Mental Health grant MH079197; National Institute of General Medicine grants GM068957, GM077183 and GM070357; and National Science Foundation grant PHY0548484. A.R. is supported by a National Science Foundation fellowship DMS-0603392 and a Burroughs Wellcome Fund Career Award at the Scientific Interface.

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Correspondence to Arjun Raj or Sanjay Tyagi.

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Supplementary Figures 1–7, Supplementary Methods (PDF 3718 kb)

Supplementary Software

Particle identification and localization software. (ZIP 3295 kb)

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Raj, A., van den Bogaard, P., Rifkin, S. et al. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5, 877–879 (2008).

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