Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Levsky, J.M. & Singer, R.H. Gene expression and the myth of the average cell. Trends Cell Biol. 13, 4–6 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Raj, A., Peskin, C.S., Tranchina, D., Vargas, D.Y. & Tyagi, S. Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 4, e309 (2006).

    Article  Google Scholar 

  3. 3

    Kaufmann, B.B. & van Oudenaarden, A. Stochastic gene expression: from single molecules to the proteome. Curr. Opin. Genet. Dev. 17, 107–112 (2007).

    CAS  Article  Google Scholar 

  4. 4

    Huang, S. Non-genetic heterogeneity of cells in development: more than just noise. Development 136, 3853–3862 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Larsson, C., Grundberg, I., Soderberg, O. & Nilsson, M. In situ detection and genotyping of individual mRNA molecules. Nat. Methods 7, 395–397 (2010).

    CAS  Article  Google Scholar 

  6. 6

    Soderberg, O. et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3, 995–1000 (2006).

    Article  Google Scholar 

  7. 7

    Jarvius, M. et al. In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method. Mol. Cell Proteomics 6, 1500–1509 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Weibrecht, I., Grundberg, I., Nilsson, M. & Soderberg, O. Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells. PLoS ONE 6, e20148 (2011).

    CAS  Article  Google Scholar 

  9. 9

    Larsson, C. et al. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nat. Methods 1, 227–232 (2004).

    CAS  Article  Google Scholar 

  10. 10

    Liu, Y. et al. Western blotting via proximity ligation for high-performance protein analysis. Mol. Cell Proteomics 10, O111.011031 (2011).

    Article  Google Scholar 

  11. 11

    Clausson, C.M. et al. Increasing the dynamic range of in situ PLA. Nat. Methods 8, 892–893 (2011).

    CAS  Article  Google Scholar 

  12. 12

    Weibrecht, I. et al. Visualising individual sequence-specific protein-DNA interactions in situ. N. Biotechnol. 29, 589–598 (2012).

    CAS  Article  Google Scholar 

  13. 13

    Shaposhnikov, S., Larsson, C., Henriksson, S., Collins, A. & Nilsson, M. Detection of Alu sequences and mtDNA in comets using padlock probes. Mutagenesis 21, 243–247 (2006).

    CAS  Article  Google Scholar 

  14. 14

    Wamsley, H.L. & Barbet, A.F. In situ detection of Anaplasma spp. by DNA target-primed rolling-circle amplification of a padlock probe and intracellular colocalization with immunofluorescently labeled host cell von Willebrand factor. J. Clin. Microbiol. 46, 2314–2319 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Andersson, C., Henriksson, S., Magnusson, K.E., Nilsson, M. & Mirazimi, A. In situ rolling circle amplification detection of Crimean Congo hemorrhagic fever virus (CCHFV) complementary and viral RNA. Virology 426, 87–92 (2012).

    CAS  Article  Google Scholar 

  16. 16

    Leuchowius, K.J., Weibrecht, I., Landegren, U., Gedda, L. & Soderberg, O. Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family. Cytometry A 75, 833–839 (2009).

    Article  Google Scholar 

  17. 17

    Zieba, A. et al. Bright-field microscopy visualization of proteins and protein complexes by in situ proximity ligation with peroxidase detection. Clin. Chem. 56, 99–110 (2010).

    CAS  Article  Google Scholar 

  18. 18

    John, H.A., Birnstiel, M.L. & Jones, K.W. RNA-DNA hybrids at the cytological level. Nature 223, 582–587 (1969).

    CAS  Article  Google Scholar 

  19. 19

    Gall, J.G. & Pardue, M.L. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. USA 63, 378–383 (1969).

    CAS  Article  Google Scholar 

  20. 20

    Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

    Emmert-Buck, M.R. et al. Laser capture microdissection. Science 274, 998–1001 (1996).

    CAS  Article  Google Scholar 

  23. 23

    Espina, V. et al. Laser-capture microdissection. Nat. Protoc. 1, 586–603 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Kenworthy, A.K. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24, 289–296 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Cremazy, F.G. et al. Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM. Exp. Cell Res. 309, 390–396 (2005).

    CAS  Article  Google Scholar 

  26. 26

    Hu, C.D., Chinenov, Y. & Kerppola, T.K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell. 9, 789–798 (2002).

    CAS  Article  Google Scholar 

  27. 27

    Soderberg, O. et al. Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45, 227–232 (2008).

    Article  Google Scholar 

  28. 28

    Leuchowius, K.J., Weibrecht, I. & Soderberg, O. In situ proximity ligation assay for microscopy and flow cytometry. Curr. Protoc. Cytom. 56, 9.36.1–9.36.15 (2011).

    Article  Google Scholar 

Download references


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).

Author information



Corresponding authors

Correspondence to Mats Nilsson or Ola Söderberg.

Ethics declarations

Competing interests

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

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing