In Situ Hybridization to Cellular RNA

Rolf Zeller1, Melissa Rogers2, Anna G. Haramis1, André s E. Carrasceo3

1 University of Utrecht, null, null, 2 University of South Florida, Tampa, Florida, 3 Buenos Aires Medical School, Buenos Aires, Argentina
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.3
DOI:  10.1002/0471142727.mb1403s55
Online Posting Date:  August, 2001
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Abstract

In situ hybridization to cellular RNA is used to determine the cellular localization of specific messages within complex cell populations and tissues. In this unit, protocols are described for hybridizing slide-mounted paraffin sections or cryosections with labeled probes. Support protocols describe synthesis of 35S-labeled riboprobes and dsDNA probes, which are then detected using film autoradiography or emulsion autoradiography. Another support protocol describes synthesis of digoxigenin-labeled RNA probes, which are non-radioactive and thus have several advantages. They are easily synthesized in large quantities, they are stable for several months, and they can be reused up to three times. An additional advantage of RNA versus DNA probes is that they result in cleaner signals because nonspecifically bound probe is removed during ribonuclease treatment.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Hybridization Using Paraffin Sections and Cells
  • Alternate Protocol: Hybridization Using Cryosections
  • Support Protocol 1: Synthesis of 35S-labeled Riboprobes
  • Support Protocol 2: Synthesis of 35S-Labeled Double-Stranded DNA Probes
  • Support Protocol 3: Synthesis of Digoxigenin-Labeled RNA Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Hybridization Using Paraffin Sections and Cells

 Materials
  • Specimens mounted on subbed glass slides (e.g., see UNIT 14.1)
  • Dewaxing/rehydration (dehydration) series consisting of 3 staining dishes of xylenes, 2 staining dishes of 100% ethanol, and 1 staining dish each of 95%, 70%, and 50% ethanol
  • 0.2 N HCl
  • 2× SSC (APPENDIX 2), 70°C
  • 1× and 3× phosphate-buffered saline (PBS; UNIT 14.1)
  • Predigested pronase solution (optional; see recipe)
  • 2 mg/ml glycine in 1× PBS (optional)
  • 4% paraformaldehyde (PFA) fixative (UNIT 14.1), freshly prepared at room temperature
  • 10 mM dithiothreitol (DTT)/1× PBS (see recipe), freshly prepared at 45°C
  • Blocking solution (see recipe), prepared immediately before use at 45°C
  • Triethanolamine (TEA) buffer (see recipe), freshly prepared
  • Acetic anhydride
  • [35S]UTP-labeled riboprobes (see Support Protocol 1)
  • S-riboprobe competitor (see recipe)
  • 50 mM DTT, sterile
  • Hybridization mix A (see recipe)
  • Moist chamber solution A (see recipe)
  • Wash solutions A, B, and C (see recipes)
  • RNase digestion solution (see recipe)
  • 50% ethanol/0.3 M ammonium acetate
  • 70% ethanol/0.3 M ammonium acetate
  • 95% ethanol/0.3 M ammonium acetate
  • 100% ethanol
  • Two sets of slide racks (one clearly labeled for RNase use only)
  • ³10 glass staining dishes
  • 45°, 55°, and 50°C water baths
  • Slide box with desiccant (e.g., Humicaps, United Desiccants–Gates)
  • 100°C heating block or water bath
  • 45°C incubator
  • Moist chambers (Fig. A.3E.4; one clearly labeled for RNase use only)
  • ³4 glass staining dishes, clearly labeled for RNase use only
  • Additional reagents and equipment for fixation and sectioning of tissues and cells (UNIT 14.1), probe preparation (see Support Protocols 1 and 2), autoradiography (UNIT 14.4), and staining and mounting (UNIT 14.5).

NOTE: All of the following steps are performed by incubating the slides containing specimens (held in a slide rack) in glass staining dishes in the indicated solutions. All solutions are made up fresh and are used only once unless indicated otherwise.

Alternate Protocol: Hybridization Using Cryosections

 Materials
  • Specimens mounted on subbed glass slides (UNIT 14.2)
  • Predigested pronase solution (see recipe)
  • 50 mM Tris×Cl, pH 7.5 (APPENDIX 2)/5 mM EDTA
  • 2 mg/ml glycine in 1× PBS
  • 1× phosphate-buffered saline (PBS; UNIT 14.1)
  • Triethanolamine (TEA) buffer (see recipe), freshly prepared
  • Acetic anhydride
  • 2× SSC (APPENDIX 2)
  • 30%, 60%, 80%, 95%, and 100% ethanol
  • Labeled DNA or RNA probe (see Support protocols 1, 2, and 3)
  • Hybridization mix B (see recipe)
  • Deionized formamide (see recipe)
  • 50% dextran sulfate (Amersham Pharmacia Biotech)
  • 3.3 M dithiothreitol (DTT), freshly prepared
  • Moist chamber solution B (see recipe)
  • DNA wash solution (see recipe), prewarmed to 37°C
  • RNA wash solutions I and II (see recipes), prewarmed to 50°C
  • 20 µg/ml boiled ribonuclease A (UNIT 7.3) in 0.5 M NaCl/10 mM Tris×Cl, pH 8.0
  • 0.6 M NaCl in 30% ethanol and in 60% ethanol
  • 2 sets of slide racks and jars (one set will be reserved for RNase use only)
  • 50°C water bath
  • Moist chamber
  • 37° or 42°C incubator (or water bath)
  • Additional reagents and equipment for fixation and sectioning of tissues (UNIT 14.2), probe preparation (see Support Protocols 1 and 2), autoradiography (UNIT 14.4), and staining and mounting (UNIT 14.5).

Support Protocol 1: Synthesis of 35S-labeled Riboprobes

 Materials
  • 5× transcription buffer (see recipe)
  • 1 M dithiothreitol (DTT), freshly prepared
  • Ribonuclease inhibitor (e.g., Amersham placental ribonuclease inhibitor or Promega Biotec RNasin)
  • 10 mM CTP, ATP, and GTP (UNIT 3.4)
  • 1 µg/µl restriction enzyme–digested plasmid (see recipe)
  • 1000 to 1500 Ci/mmol [35S]UTP (UNIT 3.4)
  • SP6 or T7 RNA polymerase (UNIT 3.8)
  • 10 mg/ml yeast tRNA or mouse poly(A) RNA for carrier
  • 1 U/µl RNase-free DNase I (e.g., Promega Biotec RQ1; UNITS 4.1 & 4.10)
  • 3 M sodium acetate
  • 7.5 M ammonium acetate
  • 100% and 70% ethanol, –20°C

NOTE: When 35S-labeled probes are prepared, it is very important to add 10 mM DTT to all solutions containing [35S]UTP, particularly after any step that may inactivate DTT (e.g. precipitation, column chromatography, and boiling). In addition, extreme care should be taken to prevent RNase contamination of reagents.

Support Protocol 3: Synthesis of Digoxigenin-Labeled RNA Probes

 Materials
  • Distilled water, sterile
  • 10× transcription buffer: 400 mM Tris×Cl (pH 8.25)/60 mM MgCl2/20 mM spermidine (Boehringer Mannheim)
  • 0.2 M dithiothreitol (DTT; APPENDIX 2)
  • Nucleotide mix, pH 8: 10 mM GTP/10 mM ATP/10 mM CTP/6.5 mM UTP/ 3.5 mM digoxigenin-UTP (Boehringer Mannheim)
  • 1 µg/ml linearized plasmid
  • 100 U/ml placental ribonuclease inhibitor (RNasin, Boehringer Mannheim)
  • 10 U/µl SP6, T3, or T7 RNA polymerase
  • TBE electrophoresis buffer (see recipe)
  • RNase-free DNase I
  • TE buffer, pH 8 (APPENDIX 2), DEPC-treated (UNIT 4.1)
  • 4 M LiCl, DEPC-treated
  • 70% ethanol in DEPC-treated water
  • 100% ethanol
  • 37°C water bath
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, 1988)
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Figures

  •  FigureFigure 14.3.1 Application of hybridization mix to slides. Carefully spread hybridization on sections with the tip of a pipet (avoid touching sections). Arrows indicate directions of spreading of hybridization mix A (or B).
    View Image

Videos

Literature Cited

Literature Cited
    Akam, M.E. 1983. The location of Ultrabithorax transcripts in Drosophila tissue sections. EMBO J. 2:2075-2084.
    Awgulewitsch, A., Utset, M.F., Hart, C.P., McGinnis, W., and Ruddle, F.H. 1986. Spatial restriction in expression of a mouse homeobox locus within the central nervous system. Nature 320:328-335.
    Brahic, M. and Haase, A.T. 1978. Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc. Natl. Acad. Sci. U.S.A. 75:6125-6129.
    Cox, K.H., DeLeon, D.V., Angerer, L.M., Angerer, R.C. 1984. Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101:485-502.
    Gall, J.G. and Pardue, M.L. 1971. Nucleic acid hybridization in cytological preparations. Meth. Enzymol. 38:470-480.
    Hafen, E., Levine, M., Garber, R.L., Gehring, W.J. 1983. An improved in situ hybridization method for the detection of cellular RNA in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia complex. EMBO J. 2:617-623.
    Haramis, A.G. and Carrasco, A.E. 1996. Whole-mount in situ hybridization and detection of RNAs in vertebrate embryos and isolated organs. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 14.9.1-14.9.18. John Wiley & Sons, New York.
    Hayashi, S., Gilam, I.C., Delaney, A.D., Tener, G.M. 1978. Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridizations with 125I-labeled RNA. J. Histochem. Cytochem. 26:677-679.
    Lawrence, J.B. and Singer, R.H. 1985. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucl. Acids Res. 13:1777-1799.
    Pardue, M.L. 1985. In situ Hybridization. In Nucleic Acid Hybridization: A Practical Approach (B.D. Hames and S.J. Higgins, eds.) pp. 179-202. IRL Press, Oxford.
    Singer, R.H., Lawrence, J.B., Villnave, C. 1986. Optimization of in situ hybridization using isotopic and nonisotopic methods. BioTechniques 4:230-250.
    Singer, R.H. and Ward, D.C. 1982. Actin gene expression visualized in a chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc. Natl. Acad. Sci. U.S.A. 79:7331-7335.
    Wilkinson, D.G. 1993. In situ Hybridization. In Essential Development Biology. A Practical Approach (C.D. Stern and P.W. H. Hollan, eds.) pp. 257-276. IRL Press, Oxford.
    Voytas, D. 1988. Agarose gel electrophoresis. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Strukl eds.) pp. 2.5.1-2.5.9. John Wiley & Sons, New York.
    Zeller, R., Bloch, K.D., William, B.S., Arceci, R.J., and Seidman, C.E. 1987. Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis. Genes Dev. 1:693-698.
 Additional References
 Chicken
    Hayashi, M., Ninomiya, Y., Parsons, J., Hayashi, K., Olsen, B.R., and Trelstad, R.L. 1986. Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes. J. Cell Biol. 102:2302-2309.
    Wedden, S.E., Pang, K., and Eichele, G. 1989. Expression pattern of homeobox-containing genes during chick embryogenesis. Development. 105:639-650.
 Drosophila
    Hafen, E. and Levine, M. 1986. The localization of RNAs in Drosophila tissue sections by in situ hybridization. In Drosophila: A Practical Approach (D.B. Roberts, ed.) pp. 139-157. IRL Press, Oxford.
 Human tissues and the use of 125I isotope
    Pfeifer-Ohlsson, S., Goustin, A.S., Rydner, J., Bjersing, L., Wahlstrom, T., Stehelin, D., Ohlsson, R. 1984. Spatial and temporal pattern of cellular myc oncogene expression in developing human placenta: Implications for embryonic cell proliferation. Cell 38:585-596.
 Nematodes
    Costa, M., Weir, M., Coulson, A., Sulston, J., and Kenyon, C. 1988. Posterior pattern formation in C. elegans involves position-specific expression of a gene containing a homeobox. Cell 55:747-756.
 Xenopus laevis
    Carrasco, A.E. and Malacinski, G.M. 1987. Localization of Xenopus homeobox gene transcripts during embryogenesis and in the adult nervous system. Dev. Biol. 121:69-73.
    Kintner, C.R. and Melton, D.A. 1987. Expression of Xenopus N-CAM RNA in ectoderm in early response to neural induction. Development 199:311-325.
 Key References
    Hogan, B., Constantini, F., Lacy, E. 1986. Manipulating the Mouse Embryo. Cold Spring Harbor, N.Y..
    Pardue et al., 1985. See above.
    Zeller et al., 1987. See above.

The Basic Protocol for Paraplast wax–embedded samples is entirely based on Zeller et al., 1987.

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