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Single-Molecule RNA In Situ Hybridization (smFISH) and Immunofluorescence (IF) in the Drosophila Egg Chamber

  • Livia V. Bayer
  • Mona Batish
  • Stephen K. Formel
  • Diana P. Bratu
Part of the Methods in Molecular Biology book series (MIMB, volume 1328)

Abstract

Detection of nucleic acids in whole tissues has become key in our understanding of gene expression during development. In situ hybridization (ISH) has been an invaluable technique in the making of numerous discoveries. Most recently, the technical advance of using short, fluorescently labeled probes has allowed for the detection of single-mRNA molecules. Thus, quantification of RNA levels in single cells or even within subcellular regions is now possible without RNA isolation. In combination with the immunofluorescence (IF) technique, visualization of nucleic acids and associating proteins is achieved with higher resolution than ever before using light microscopy. Here we describe the steps implemented to achieve the visualization of individual messenger RNAs (mRNA) using single-molecule FISH (smFISH) probes, as well as detection of mRNA/protein (mRNP) complexes via smFISH in combination with IF.

Keywords

mRNA mRNP Stellaris™ probe smFISH Immunofluorescence Egg chambers Oogenesis Drosophila melanogaster 

Notes

Acknowledgements

We would like to thank the members of the Bratu lab for constructive criticism during the preparation of this manuscript. LVB, SKF and DPB were supported by NSF CAREER award to DPB. The Gurken 1D12 antibody, developed at the California Institute of Technology, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA.

References

  1. 1.
    Gall JG, Pardue ML (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A 63:378–383PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Wetmur JG (1991) DNA probes: applications of the principles of nucleic acid hybridization. Crit Rev Biochem Mol Biol 26:227–259CrossRefPubMedGoogle Scholar
  3. 3.
    Boutorine AS, Novopashina DS, Krasheninina OA et al (2013) Fluorescent probes for nucleic Acid visualization in fixed and live cells. Molecules 18:15357–15397CrossRefPubMedGoogle Scholar
  4. 4.
    Beaucage SL, Iyer RP (1992) Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach. Tetrahedron 48:2223–2311CrossRefGoogle Scholar
  5. 5.
    Roget A, Bazin H, Teoule R (1989) Synthesis and use of labelled nucleoside phosphoramidite building blocks bearing a reporter group: biotinyl, dinitrophenyl, pyrenyl and dansyl. Nucleic Acids Res 17:7643–7651PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Tyagi S, Bratu DP, Kramer FR (1998) Multicolor molecular beacons for allele discrimination. Nat Biotechnol 16:49–53CrossRefPubMedGoogle Scholar
  7. 7.
    Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308CrossRefPubMedGoogle Scholar
  8. 8.
    Bauman JG, Wiegant J, Borst P et al (1980) A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome-labelled RNA. Exp Cell Res 128:485–490CrossRefPubMedGoogle Scholar
  9. 9.
    Raap AK, van de Corput MP, Vervenne RA et al (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum Mol Genet 4:529–534CrossRefPubMedGoogle Scholar
  10. 10.
    Femino AM, Fay FS, Fogarty K et al (1998) Visualization of single RNA transcripts in situ. Science 280:585CrossRefPubMedGoogle Scholar
  11. 11.
    Raj A, van den Bogaard P, Rifkin SA et al (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5:877–879PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Raj A, Tyagi S (2010) Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol 472:365–386CrossRefPubMedGoogle Scholar
  13. 13.
    Batish M, Raj A, Tyagi S (2011) Single molecule imaging of RNA in situ. Methods Mol Biol 714:3–13CrossRefPubMedGoogle Scholar
  14. 14.
    Little SC, Tkacik G, Kneeland TB et al (2011) The formation of the Bicoid morphogen gradient requires protein movement from anteriorly localized mRNA. PLoS Biol 9:e1000596PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Preibisch S, Saalfeld S, Tomancak P (2009) Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25:1463–1465PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Livia V. Bayer
    • 1
    • 2
  • Mona Batish
    • 3
  • Stephen K. Formel
    • 1
  • Diana P. Bratu
    • 4
    • 5
  1. 1.Department of Biological SciencesHunter College of the City University of New YorkNew YorkUSA
  2. 2.Molecular, Cellular, and Developmental Biology ProgramThe Graduate Center, CUNYNew YorkUSA
  3. 3.Department of Microbiology and Molecular GeneticsRutgers New Jersey Medical SchoolNewarkUSA
  4. 4.Department of Biological SciencesHunter College of the City University of New YorkNew YorkUSA
  5. 5.Molecular, Cellular, and Developmental Biology ProgramThe Graduate Center, CUNYNew YorkUSA

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