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In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay

Nature Protocols volume 8, pages 355–372 (2013)Cite this article

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Abstract

Analysis at the single-cell level is essential for the understanding of cellular responses in heterogeneous cell populations, but it has been difficult to perform because of the strict requirements put on detection methods with regard to selectivity and sensitivity (i.e., owing to the cross-reactivity of probes and limited signal amplification). Here we describe a 1.5-d protocol for enumerating and genotyping mRNA molecules in situ while simultaneously obtaining information on protein interactions or post-translational modifications; this is achieved by combining padlock probes with in situ proximity ligation assays (in situ PLA). In addition, we provide an example of how to design padlock probes and how to optimize staining conditions for fixed cells and tissue sections. Both padlock probes and in situ PLA provide the ability to directly visualize single molecules by standard microscopy in fixed cells or tissue sections, and these methods may thus be valuable for both research and diagnostic purposes.

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Figure 1: Scheme of the combined mRNA and protein detection protocol.
Figure 2: Design of the padlock probe and LNA primer for the ACTB mRNA (encoding β-actin) as an example.
Figure 3: Example image of combined padlock and in situ PLA staining.
Figure 4: Example of combined padlock and in situ PLA staining for phosphorylated PDGFR-β and DUSP6 mRNA.
Figure 5: Example of detection of ACTB in FFPE tissue section.

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Acknowledgements

This work was supported by grants from the Swedish Research Council, the Wallenberg Foundation and the EU FP7 projects 278568 (PRIMES), 259796 (DIATOOLS) and 201418 (READNA). B.K. is supported by the Deutsche Forschungsgemeinschaft (Ko 4345/1-1).

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Author notes

  1. Irene Weibrecht, Ida Grundberg & Mats Nilsson

    Present address: Present addresses: Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany (I.W.); Olink Bioscience, Uppsala Science Park, Uppsala, Sweden (I.G.); Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden (M.N.).,

  2. Irene Weibrecht, Elin Lundin, Mats Nilsson and Ola Söderberg: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology, Science for Life Laboratory, Rudbeck Laboratory, Genetics and Pathology, Uppsala University, Uppsala, Sweden

    Irene Weibrecht, Elin Lundin, Sara Kiflemariam, Marco Mignardi, Ida Grundberg, Chatarina Larsson, Björn Koos, Mats Nilsson & Ola Söderberg

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Correspondence to Mats Nilsson or Ola Söderberg.

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Competing interests

M.N. holds stock in Olink Bioscience, which holds the commercial rights to the padlock and proximity ligation assays.

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Weibrecht, I., Lundin, E., Kiflemariam, S. et al. In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay. Nat Protoc 8, 355–372 (2013). https://doi.org/10.1038/nprot.2013.006

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