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Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos

Nature Protocols volume 4, pages 605–618 (2009)Cite this article

Abstract

We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in a 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into antisense RNA probes by in vitro transcription. The quality of each probe is evaluated before ISH using a RNA probe quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4 °C for later analysis. Starting with fixed, staged embryos, the protocol takes 6 d from probe template production through hybridization. Preparation of fixed embryos requires a minimum of 2 weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6,000 genes throughout embryogenesis.

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Figure 1: Flowchart for high-throughput RNA in situ hybridization (ISH) to whole-mount Drosophila embryos.
Figure 2: Size verification of PCR products for probe templates.
Figure 3: Quantification of RNA probes.
Figure 4: Anticipated results.

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References

  1. Gall, J.G. & Pardue, M.L. Formation and detection of RNA–DNA hybrid molecules in cytological preparations. Proc. Natl Acad. Sci. USA 63, 378–783 (1969).

    Article  CAS  Google Scholar 

  2. Jones, K.W. & Robertson, F.W. Localisation of reiterated nucleotide sequences in Drosophila and mouse by in situ hybridisation of complementary RNA. Chromosoma 31, 331–345 (1970).

    Article  CAS  Google Scholar 

  3. Harrison, P.R., Conkie, D., Paul, J. & Jones, K. Localisation of cellular globin messenger RNA by in situ hybridisation to complementary DNA. FEBS Lett. 32, 109–112 (1973).

    Article  CAS  Google Scholar 

  4. Hafen, E., Levine, M., Garber, R. & Gehring, W. An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex. EMBO J. 2, 617–623 (1983).

    Article  CAS  Google Scholar 

  5. Akam, M.E. The location of Ultrabithorax transcripts in Drosophila tissue sections. EMBO J. 2, 2075–2084 (1983).

    Article  CAS  Google Scholar 

  6. Cowman, A.F., Zuker, C.S. & Rubin, G.M. An opsin gene expressed in only one photoreceptor cell type of the Drosophila eye. Cell 44, 705–710 (1986).

    Article  CAS  Google Scholar 

  7. Banerjee, U., Renfranz, P.J., Pollock, J.A. & Benzer, S. Molecular characterization and expression of sevenless, a gene involved in neuronal pattern formation in the Drosophila eye. Cell 49, 281–291 (1987).

    Article  CAS  Google Scholar 

  8. 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 

  9. Lehmann, R. & Tautz, D. In situ hybridization to RNA. Methods Cell Biol. 44, 575–598 (1994).

    Article  CAS  Google Scholar 

  10. Kopczynski, C.C. et al. A high throughput screen to identify secreted and transmembrane proteins involved in Drosophila embryogenesis. Proc. Natl Acad. Sci. USA 95, 9973–9978 (1998).

    Article  CAS  Google Scholar 

  11. Simin, K. et al. Profiling patterned transcripts in Drosophila embryos. Genome Res. 12, 1040–1047 (2002).

    Article  CAS  Google Scholar 

  12. Tomancak, P. et al. Systematic determination of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 3, RESEARCH0088 (2002).

  13. Tomancak, P. et al. Global analysis of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 8, R145 (2007).

    Article  Google Scholar 

  14. Rubin, G.M. et al. A Drosophila complementary DNA resource. Science 287, 2222–2224 (2000).

    Article  CAS  Google Scholar 

  15. Stapleton, M. et al. The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes. Genome Res. 12, 1294–1300 (2002).

    Article  Google Scholar 

  16. Stapleton, M. et al. A Drosophila full-length cDNA resource. Genome Biol. 3, research0080 (2002).

  17. Eugene, O., Yund, A. & Fristrom, J.W. Preparative isolation and short term organ culture of imaginal discs of Drosophila melanogaster . Tissue Culture Assoc. Manual 5, 1055–1062 (1979).

    Article  Google Scholar 

  18. Kornberg, T., Siden, I., O'Farrell, P. & Simon, M. The engrailed locus of Drosophila: in situ localization of transcripts reveals compartment-specific expression. Cell 40, 45–53 (1985).

    Article  CAS  Google Scholar 

  19. Schroder, C., Tautz, D., Seifert, E. & Jackle, H. Differential regulation of the two transcripts from the Drosophila gap segmentation gene hunchback. EMBO J. 7, 2881–2887 (1988).

    Article  CAS  Google Scholar 

  20. Jazwinska, A., Kirov, N., Wieschaus, E., Roth, S. & Rushlow, C. The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation. Cell 96, 563–573 (1999).

    Article  CAS  Google Scholar 

  21. Pfeiffer, B.D. et al. Tools for neuroanatomy and neurogenetics in Drosophila . Proc. Natl. Acad. Sci. USA 105, 9715–9720 (2008).

    Article  CAS  Google Scholar 

  22. Harmon, C.L. et al. Comparative analysis of spatial patterns of gene expression in Drosophila melanogaster imaginal discs. In Research in Computational Molecular Biology: Proc. 11th Annual Intl. Conf. (RECOMB 2007), Lecture Notes in Bioinformatics 4453 (Eds. Speed, T. and Huang, H.), 533–547 (Springer-Verlag, Berlin, Germany, 2007).

    Chapter  Google Scholar 

  23. Lecuyer, E. et al. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131, 174–187 (2007).

    Article  CAS  Google Scholar 

  24. Fowlkes, C.C. et al. A quantitative spatiotemporal atlas of gene expression in the Drosophila blastoderm. Cell 133, 364–774 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank current and former members of the BDGP, especially Amy Beaton, Joe Carlson, Erwin Frise, Elaine Kwan, Todd Laverty, Barret Pfeiffer, Stephen Richards, Mark Stapleton, Pavel Tomancak and Gerry Rubin for their contributions to these protocols as part of the systematic determination of patterns of gene expression in the Drosophila embryogenesis project. We thank Ken Wan for critical comments on the manuscript. This work was supported by the NIH Grant R01 GM076655 (SEC) through the US Department of Energy under contract no. DE-AC02-05CH11231.

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  1. Life Sciences Division, Berkeley Drosophila Genome Project, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Richard Weiszmann, Ann S Hammonds & Susan E Celniker

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  1. Richard Weiszmann
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  2. Ann S Hammonds
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  3. Susan E Celniker
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Correspondence to Ann S Hammonds.

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Weiszmann, R., Hammonds, A. & Celniker, S. Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos. Nat Protoc 4, 605–618 (2009). https://doi.org/10.1038/nprot.2009.55

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