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Validating transcripts with probes and imaging technology

Nature Methods volume 8, pages S12–S19 (2011)Cite this article

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Abstract

High-throughput gene expression screens provide a quantitative picture of the average expression signature of biological samples. However, the analysis of spatial gene expression patterns with single-cell resolution requires quantitative in situ measurement techniques. Here we describe recent technological advances in RNA fluorescence in situ hybridization (FISH) techniques that facilitate detection of individual fluorescently labeled mRNA molecules of practically any endogenous gene. These methods, which are based on advances in probe design, imaging technology and image processing, enable the absolute measurement of transcript abundance in individual cells with single-molecule resolution.

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Figure 1: Common steps in ISH.
Figure 2: Probe designs for ISH.

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Acknowledgements

We thank S. Semrau, J.P. Junker, S. Mukherji and A. Lavi-Itzkovitz for valuable comments. This work was supported by the US National Institutes of Health National Cancer Institute Physical Sciences Oncology Center at the Massachusetts Institute of Technology (U54CA143874) and a US National Institutes of Health Pioneer award (1DP1OD003936) to A.v.O.; S.I. acknowledges support from the European Molecular Biology Organization, the Human Frontiers Science Program and the Machiah Foundation.

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  1. Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Shalev Itzkovitz & Alexander van Oudenaarden

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Shalev Itzkovitz & Alexander van Oudenaarden

  3. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Alexander van Oudenaarden

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Correspondence to Alexander van Oudenaarden.

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Itzkovitz, S., van Oudenaarden, A. Validating transcripts with probes and imaging technology. Nat Methods 8 (Suppl 4), S12–S19 (2011). https://doi.org/10.1038/nmeth.1573

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