Skip to main content

Thank you for visiting nature.com. 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.

  • Review Article
  • Published:

Validating transcripts with probes and imaging technology

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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

Similar content being viewed by others

References

  1. Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

    Article  CAS  Google Scholar 

  2. Heller, R.A. et al. Discovery and analysis of inflammatory disease-related genes using cDNA microarrays. Proc. Natl. Acad. Sci. USA 94, 2150–2155 (1997).

    Article  CAS  Google Scholar 

  3. Sorlie, T. et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc. Natl. Acad. Sci. USA 98, 10869–10874 (2001).

    Article  CAS  Google Scholar 

  4. van't Veer, L.J. et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 415, 530–536 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Raj, A. & van Oudenaarden, A. Nature, nurture or chance: stochastic gene expression and its consequences. Cell 135, 216–226 (2008).

    Article  CAS  Google Scholar 

  7. Lengauer, C., Kinzler, K.W. & Vogelstein, B. Genetic instability in colorectal cancers. Nature 386, 623–627 (1997).

    Article  CAS  Google Scholar 

  8. Park, S.Y., Gonen, M., Kim, H.J., Michor, F. & Polyak, K. Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype. J. Clin. Invest. 120, 636–644 (2010).

    Article  CAS  Google Scholar 

  9. Navin, N. et al. Inferring tumor progression from genomic heterogeneity. Genome Res. 20, 68–80 (2010).

    Article  CAS  Google Scholar 

  10. Reya, T., Morrison, S.J., Clarke, M.F. & Weissman, I.L. Stem cells, cancer and cancer stem cells. Nature 414, 105–111 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Freeman, W.M., Walker, S.J. & Vrana, K.E. Quantitative RT-PCR: pitfalls and potential. Biotechniques 26, 112–125 (1999).

    Article  CAS  Google Scholar 

  13. Warren, L., Bryder, D., Weissman, I.L. & Quake, S.R. Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc. Natl. Acad. Sci. USA 103, 17807–17812 (2006).

    Article  CAS  Google Scholar 

  14. van der Flier, L.G. et al. Transcription factor achaete scute-like 2 controls intestinal stem cell fate. Cell 136, 903–912 (2009).

    Article  CAS  Google Scholar 

  15. Levsky, J.M. & Singer, R.H. Fluorescence in situ hybridization: past, present and future. J. Cell Sci. 116, 2833–2838 (2003).

    Article  CAS  Google Scholar 

  16. van der Ploeg, M. Cytochemical nucleic acid research during the twentieth century. Eur. J. Histochem. 44, 7–42 (2000).

    CAS  PubMed  Google Scholar 

  17. Gregorieff, A. & Clevers, H. In situ hybridization to identify gut stem cells. Curr. Protoc. Stem. Cell. Biol. chapter 11, unit 2F.1 (2010).

  18. Pare, A. et al. Visualization of individual Scr mRNAs during Drosophila embryogenesis yields evidence for transcriptional bursting. Curr. Biol. 19, 2037–2042 (2009).

    Article  CAS  Google Scholar 

  19. Randolph, J.B. & Waggoner, A.S. Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes. Nucleic Acids Res. 25, 2923–2929 (1997).

    Article  CAS  Google Scholar 

  20. Larson, D.R., Singer, R.H. & Zenklusen, D. A single molecule view of gene expression. Trends Cell Biol. 19, 630–637 (2009).

    Article  CAS  Google Scholar 

  21. Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA transcripts in situ . Science 280, 585–590 (1998). This pioneering work demonstrated single-molecule transcript imaging in fixed cells using multiply-labeled fluorescent probes.

    Article  CAS  Google Scholar 

  22. Femino, A.M., Fogarty, K., Lifshitz, L.M., Carrington, W. & Singer, R.H. Visualization of single molecules of mRNA in situ . Methods Enzymol. 361, 245–304 (2003).

    Article  CAS  Google Scholar 

  23. Maamar, H., Raj, A. & Dubnau, D. Noise in gene expression determines cell fate in Bacillus subtilis . Science 317, 526–529 (2007).

    Article  CAS  Google Scholar 

  24. Zenklusen, D., Larson, D.R. & Singer, R.H. Single-RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15, 1263–1271 (2008).

    Article  CAS  Google Scholar 

  25. Tan, R.Z. & van Oudenaarden, A. Transcript counting in single cells reveals dynamics of rDNA transcription. Mol. Syst. Biol. 6, 358 (2010).

    Article  Google Scholar 

  26. 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 

  27. Capodieci, P. et al. Gene expression profiling in single cells within tissue. Nat. Methods 2, 663–665 (2005).

    Article  CAS  Google Scholar 

  28. 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). Single-molecule transcript imaging in cells and in fixed tissues was achieved using multiple singly labeled probes.

    Article  CAS  Google Scholar 

  29. To, T.L. & Maheshri, N. Noise can induce bimodality in positive transcriptional feedback loops without bistability. Science 327, 1142–1145 (2010).

    Article  CAS  Google Scholar 

  30. Khalil, A.M. et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl. Acad. Sci. USA 106, 11667–11672 (2009).

    Article  CAS  Google Scholar 

  31. Raj, A., Rifkin, S.A., Andersen, E. & van Oudenaarden, A. Variability in gene expression underlies incomplete penetrance. Nature 463, 913–918 (2010).

    Article  CAS  Google Scholar 

  32. Taniguchi, Y. et al. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329, 533–538 (2010).

    Article  CAS  Google Scholar 

  33. Lu, J. & Tsourkas, A. Imaging individual microRNAs in single mammalian cells in situ . Nucleic Acids Res. 37, e100 (2009).

    Article  Google Scholar 

  34. Larsson, C., Grundberg, I., Soderberg, O. & Nilsson, M. In situ detection and genotyping of individual mRNA molecules. Nat. Methods 7, 395–397 (2010). This work introduced a sensitive single-molecule transcript-imaging technique that uses padlock probes and in situ target-primed rolling-circle amplification in cells and tissues to specifically detect targets with a single nucleotide difference.

    Article  CAS  Google Scholar 

  35. Wittung, P., Nielsen, P.E., Buchardt, O., Egholm, M. & Norden, B. DNA-like double helix formed by peptide nucleic acid. Nature 368, 561–563 (1994).

    Article  CAS  Google Scholar 

  36. Lansdorp, P.M. et al. Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet. 5, 685–691 (1996).

    Article  CAS  Google Scholar 

  37. Kloosterman, W.P., Wienholds, E., de Bruijn, E., Kauppinen, S. & Plasterk, R.H. In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat. Methods 3, 27–29 (2006).

    Article  CAS  Google Scholar 

  38. Player, A.N., Shen, L.P., Kenny, D., Antao, V.P. & Kolberg, J.A. Single-copy gene detection using branched DNA (bDNA) in situ hybridization. J. Histochem. Cytochem. 49, 603–612 (2001).

    Article  CAS  Google Scholar 

  39. Levsky, J.M., Shenoy, S.M., Pezo, R.C. & Singer, R.H. Single-cell gene-expression profiling. Science 297, 836–840 (2002). This work substantially increased the number of targets that can be simultaneously imaged by using combination of probes labeled with spectrally distinct fluorophores.

    Article  CAS  Google Scholar 

  40. Nederlof, P.M., van der Flier, S., Vrolijk, J., Tanke, H.J. & Raap, A.K. Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy. Cytometry 13, 839–845 (1992).

    Article  CAS  Google Scholar 

  41. Nederlof, P.M. et al. Multiple fluorescence in situ hybridization. Cytometry 11, 126–131 (1990).

    Article  CAS  Google Scholar 

  42. Park, H.Y., Buxbaum, A.R. & Singer, R.H. Single mRNA tracking in live cells. Methods Enzymol. 472, 387–406 (2010).

    Article  CAS  Google Scholar 

  43. Golding, I., Paulsson, J., Zawilski, S.M. & Cox, E.C. Real-time kinetics of gene activity in individual bacteria. Cell 123, 1025–1036 (2005).

    Article  CAS  Google Scholar 

  44. Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998). This work introduced the MS2 method for single mRNA detection in live cells.

    Article  CAS  Google Scholar 

  45. Fusco, D. et al. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr. Biol. 13, 161–167 (2003).

    Article  CAS  Google Scholar 

  46. Shav-Tal, Y. et al. Dynamics of single mRNPs in nuclei of living cells. Science 304, 1797–1800 (2004).

    Article  CAS  Google Scholar 

  47. Haim, L., Zipor, G., Aronov, S. & Gerst, J.E. A genomic integration method to visualize localization of endogenous mRNAs in living yeast. Nat. Methods 4, 409–412 (2007).

    Article  CAS  Google Scholar 

  48. Rackham, O. & Brown, C.M. Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs. EMBO J. 23, 3346–3355 (2004).

    Article  CAS  Google Scholar 

  49. Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nat. Methods 6, 331–338 (2009).

    Article  CAS  Google Scholar 

  50. Valencia-Burton, M., McCullough, R.M., Cantor, C.R. & Broude, N.E. RNA visualization in live bacterial cells using fluorescent protein complementation. Nat. Methods 4, 421–427 (2007).

    Article  CAS  Google Scholar 

  51. Ozawa, T., Natori, Y., Sato, M. & Umezawa, Y. Imaging dynamics of endogenous mitochondrial RNA in single living cells. Nat. Methods 4, 413–419 (2007).

    Article  CAS  Google Scholar 

  52. Tyagi, S. & Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996). Molecular beacons were introduced for the detection of single mRNAs in live cells.

    Article  CAS  Google Scholar 

  53. Vargas, D.Y., Raj, A., Marras, S.A., Kramer, F.R. & Tyagi, S. Mechanism of mRNA transport in the nucleus. Proc. Natl. Acad. Sci. USA 102, 17008–17013 (2005).

    Article  CAS  Google Scholar 

  54. Bratu, D.P., Cha, B.J., Mhlanga, M.M., Kramer, F.R. & Tyagi, S. Visualizing the distribution and transport of mRNAs in living cells. Proc. Natl. Acad. Sci. USA 100, 13308–13313 (2003).

    Article  CAS  Google Scholar 

  55. Lifland, A.W., Zurla, C. & Santangelo, P.J. Single molecule densitive multivalent polyethylene glycol probes for RNA imaging. Bioconjug. Chem. 21, 483–488 (2010).

    Article  CAS  Google Scholar 

  56. Santangelo, P.J. et al. Single molecule-sensitive probes for imaging RNA in live cells. Nat. Methods 6, 347–349 (2009).

    Article  CAS  Google Scholar 

  57. Thompson, R.E., Larson, D.R. & Webb, W.W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    Article  CAS  Google Scholar 

  58. Grunwald, D., Singer, R.H. & Czaplinski, K. Cell biology of mRNA decay. Methods Enzymol. 448, 553–577 (2008).

    Article  CAS  Google Scholar 

  59. McNally, J.G., Karpova, T., Cooper, J. & Conchello, J.A. Three-dimensional imaging by deconvolution microscopy. Methods 19, 373–385 (1999).

    Article  CAS  Google Scholar 

  60. Smith, C.S., Joseph, N., Rieger, B. & Lidke, K.A. Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat. Methods 7, 373–375 (2010).

    Article  CAS  Google Scholar 

  61. van der Flier, L.G. & Clevers, H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu. Rev. Physiol. 71, 241–260 (2009).

    Article  CAS  Google Scholar 

  62. Alon, U. Network motifs: theory and experimental approaches. Nat. Rev. Genet. 8, 450–461 (2007).

    Article  CAS  Google Scholar 

  63. Bassell, G. & Singer, R.H. mRNA and cytoskeletal filaments. Curr. Opin. Cell Biol. 9, 109–115 (1997).

    Article  CAS  Google Scholar 

  64. Klar, T.A., Jakobs, S., Dyba, M., Egner, A. & Hell, S.W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).

    Article  CAS  Google Scholar 

  65. Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  66. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  67. Hell, S.W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    Article  CAS  Google Scholar 

  68. Shroff, H., Galbraith, C.G., Galbraith, J.A. & Betzig, E. Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat. Methods 5, 417–423 (2008).

    Article  CAS  Google Scholar 

  69. Bassell, G.J. et al. Sorting of beta-actin mRNA and protein to neurites and growth cones in culture. J. Neurosci. 18, 251–265 (1998).

    Article  CAS  Google Scholar 

  70. Choi, Y. et al. In situ visualization of gene expression using polymer-coated quantum-dot-DNA conjugates. Small 5, 2085–2091 (2009).

    Article  CAS  Google Scholar 

  71. Chan, P., Yuen, T., Ruf, F., Gonzalez-Maeso, J. & Sealfon, S.C. Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization. Nucleic Acids Res. 33, e161 (2005).

    Article  Google Scholar 

  72. Gonzalez-Maeso, J. et al. Identification of a serotonin/glutamate receptor complex implicated in psychosis. Nature 452, 93–97 (2008).

    Article  CAS  Google Scholar 

  73. Smith, A.M., Duan, H., Mohs, A.M. & Nie, S. Bioconjugated quantum dots for in vivo molecular and cellular imaging. Adv. Drug Deliv. Rev. 60, 1226–1240 (2008).

    Article  CAS  Google Scholar 

  74. Tholouli, E. et al. Imaging of multiple mRNA targets using quantum dot based in situ hybridization and spectral deconvolution in clinical biopsies. Biochem. Biophys. Res. Commun. 348, 628–636 (2006).

    Article  CAS  Google Scholar 

  75. Ishihama, Y. & Funatsu, T. Single molecule tracking of quantum dot-labeled mRNAs in a cell nucleus. Biochem. Biophys. Res. Commun. 381, 33–38 (2009).

    Article  CAS  Google Scholar 

  76. Weil, T.T., Parton, R.M. & Davis, I. Making the message clear: visualizing mRNA localization. Trends Cell Biol. 20, 380–390 (2010).

    Article  CAS  Google Scholar 

  77. Tautz, D. & Pfeifle, C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81–85 (1989).

    Article  CAS  Google Scholar 

  78. Bloom, K.S. et al. Using green fluorescent protein fusion proteins to quantitate microtubule and spindle dynamics in budding yeast. Methods Cell Biol. 61, 369–383 (1999).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  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

Authors
  1. Shalev Itzkovitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexander van Oudenaarden
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alexander van Oudenaarden.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1573

This article is cited by

Search

Advanced search

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