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In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples

Nature Protocols volume 1, pages 1939–1946 (2006)Cite this article

Abstract

High throughput microarray transcription analyses provide us with the expression profiles for large amounts of plant genes. However, their tissue and cellular resolution is limited. Thus, for detailed functional analysis, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here, we present an in situ mRNA hybridization method that is routinely used for the analysis of plant gene expression patterns. The protocol is optimized for whole mount mRNA localizations in Arabidopsis seedling tissues including embryos, roots, hypocotyls and young primary leaves. It can also be used for comparable tissues in other species. Part of the protocol can also be automated and performed by a liquid handling robot. Here we present a detailed protocol, recommended controls and troubleshooting, along with examples of several applications. The total time to carry out the entire procedure is ∼7 d, depending on the tissue used.

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Figure 1: Examples of various whole-mount in situ mRNA localizations in different plant tissues.

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References

  1. 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 

  2. de Almeid Engler, J., Van Montagu, M. & Engler, G. Whole-mount in situ hybridization in plants. Methods Mol. Biol. 82, 373–384 (1998).

    Google Scholar 

  3. Bennett, M.J. et al. Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism. Science 273, 948–950 (1996).

    Article  CAS  Google Scholar 

  4. Ludevid, D., Hofte, H., Himelblau, E. & Chrispeels, M.J. The expression pattern of the tonoplast intrinsic protein gamma-TIP in Arabidopsis thaliana is correlated with cell enlargement. Plant Physiol. 100, 1633–1639 (1992).

    Article  CAS  Google Scholar 

  5. Plickert, G., Gajewski, M., Gehrke, G., Gausepohl, H., Schlossherr, J. & Ibrahim, H. Automated in situ detection (AISD) of biomolecules. Dev. Genes Evol. 207, 362–367 (1997).

    Article  CAS  Google Scholar 

  6. Nonradioactive In Situ hybridization Application Manual, 2nd edition, 51–56 (Boehringer Manheim 1996).

  7. Friml, J., Benková, E., Mayer, U., Palme, K. & Muster, G. Automated whole-mount localization techniques for plant seedlings. Plant J. 34, 115–124 (2003).

    Article  CAS  Google Scholar 

  8. García-Aguilar, M., Dorantes-Acosta, A., Pérez-España, V. & Vielle-Calzada, J.P. Whole-mount in situ mRNA localization in developing ovules and seeds of Arabidopsis. Plant Mol. Biol. Reporter 23, 279–289 (2005).

    Article  Google Scholar 

  9. Zachgo, S., Perbal, M.C., Saedler, H. & Schwarz-Sommer, Z. In situ analysis of RNA and protein expression in whole mounts facilitates detection of floral gene expression dynamics. Plant J. 23, 697–702 (2000).

    Article  CAS  Google Scholar 

  10. Fisher, H.W. & Williams, R.C. Electron microscopic visualization of nucleic acids and of their complexes with proteins. Annu. Rev. Biochem. 48, 649–679 (1979).

    Article  CAS  Google Scholar 

  11. Malamy, J.E. & Benfey, P.N. Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development, 124, 33–44 (1997).

    CAS  PubMed  Google Scholar 

  12. Hamann, T., Benková, E., Bäurle, I., Kientz, M. & Jürgens, G. The Arabidopsis BODENLOS gene encodes an auxin response protein inhibiting MONOPTEROS-mediated embryo patterning. Genes & Dev. 16, 1610–1615 (2002).

    Article  CAS  Google Scholar 

  13. Friml, J. et al. AtPIN4 mediates sink driven auxin gradients and patterning in Arabidopsis roots. Cell, 108, 661–673 (2002).

    Article  CAS  Google Scholar 

  14. Blilou, I. et al. The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature, 433, 39–44 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

J.H. was supported by the Ministry of Education of the Czech Republic (LC06034, MSM0021622415); P.B. and J.F. by the VolkswagenStiftung and the EMBO Young Investigator Program; E.B. by the Margarete von Wrangell-Habilitations program.

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Authors and Affiliations

  1. Department of Functional Genomics and Proteomics, Masaryk University, Laboratory of Molecular Plant Physiology, Kamenice 5, Brno, 625 00, Czech Republic

    Jan Hejátko & Jiří Friml

  2. Department of Molecular Genetics, Utrecht University, Utrecht, 3584 CH, The Netherlands

    Ikram Blilou & Ben Scheres

  3. Centre for Plant Molecular Biology, Tübingen University, Tübingen, 72076, Germany

    Philip B Brewer, Jiří Friml & Eva Benková

Authors
  1. Jan Hejátko
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  2. Ikram Blilou
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  4. Jiří Friml
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  5. Ben Scheres
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  6. Eva Benková
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Correspondence to Eva Benková.

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Hejátko, J., Blilou, I., Brewer, P. et al. In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples. Nat Protoc 1, 1939–1946 (2006). https://doi.org/10.1038/nprot.2006.333

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