Abstract
Protocols for in situ hybridization (ISH) of cultured cells often include storage in alcohol at −20°C between fixation of the cultures and the ISH procedure. In experiments aimed at localizing ferritin mRNA in C2 muscle cultures by ISH with digoxigenin-labelled riboprobes, we have noticed that omission of the ethanol storage dramatically changed the pattern of mRNA localization. In cultures stored in 50%, 70%, or 90% ethanol for at least 15 min, ferritin signal was stronger on myotubes than myoblasts but was uniformly distributed over both. In untreated cultures, the signal was patchy, concentrated on the extremities of the elongated myoblasts and very sparse in myotubes. Similar results were obtained with a probe to β-actin used as a control, except that signal was higher in myoblasts in all conditions. When the probes were reduced in size to <100 bases from 561 for ferritin and 1150 for actin, the pattern became uniform, regardless of prehybridization treatment. The patchy pattern disappeared when cells were treated with RNase A following hybridization, suggesting that it is non-specific, despite its absence in cultures hybridized with a sense probe. We conclude that incomplete access of RNA probes can result not only in a reduced ISH signal but also in artefactual patterns of mRNA localization.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alonso, S., Minty, A., Bourlet, Y. & Buckingham, M. (1986) Comparison of three actin-coding sequences in the mouse; evolutionary relationship between the actin genes of warm-blooded vertebrates. Journal of Molecular Evolution 23, 11- 22.
Angerer, L. M. & Angerer, R. C. (1981) Detection of poly A+RNA in sea urchin eggs and embryos by quantitative in situ hybridization. Nucleic Acids Research 9, 2819- 40.
Angerer, L. M. & Angerer, R. C. (1993) In situ hybridization to cellular RNA with radiolabelled RNA probes. In: In Situ Hybridization, A Practical Approach (edited by Wilkinson, D. G. ) pp. 15- 32. Oxford: IRL Press at Oxford University Press.
Brahic, M. & Haase, A. T. (1978) Detection of viral sequences of low reiteration frequency by in situ hybridization. Proceedings of the National Academy of Sciences of the United States of America 75, 6125- 9.
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. Developmental Biology 101, 485- 502.
Cripe, L., Morris, E. & Fulton, A.B. (1993) Vimentinm RNA location changes during muscle development. Proceedings of the National Academy of Sciences of the United States of America 90, 2724- 8.
Deschepper, C. F., Mellon, S. H., Reudelhuber, T. L., Gardner, D. G., Jen, J. & Lau, Y.-F. (1988) In situ hybridization histochemistry on mRNA in endocrine tissues. In: Endocrine Genes: Analytical Methods, Experimental Approaches, and Selected Systems. New York: Oxford University Press.
Godard, C. & Jones, K.W. (1979) Detection of AKR MuLV-specific RNA in AKR mouse cells by in situ hybridization. Nucleic Acids Research 6, 2849- 61.
Goldenthal, K. L., Hedman, K., Chen, J. W., August, J. T. & Willingham, M. C. (1985) Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins. Journal of Histochemistry and Cytochemistry 33, 813- 20.
Harford, J. B. (1993) Iron regulation of transferrin receptor mRNA stability. In: Control of Messenger RNA Stability (edited by Belasco, J. G. & Brawerman, G.) pp. 239- 66. San Diego: Academic Press
Hentze, N. W. & Kuhn, L. C. (1997) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proceedings of the National Academy of Sciences of the United States of America 93, 8175- 82.
Hill, M. A., Schedlich, L. & Gunning, P. (1994) Serum-induced signal transduction determines the peripheral location of β-actin mRNA within the cell. Journal of Cell Biology 126, 1221- 9.
Kaech, S., Kim, J.B., Cariola, M. & Ralston, E. (1996) Improved lipid-mediated gene transfer into primary cultures of hippocampal neurons. Molecular Brain Research 35, 344- 8.
Kislaukis, E.H., Li, Z., Singer, R. H. & Taneja, K. L. (1993) Isoform-specific 3′-untranslated sequences sort α-cardiac and β-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. Journal of Cell Biology 123, 165- 72.
Lawrence, J. B. & Singer, R. H. (1985) Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Research 13, 1777- 99.
Llewellyn-Smith, I. J. & Minson, J. B. (1992) Complete penetration of antibodies into vibratome sections after glutaraldehyde fixation and ethanol treatment: light and electron microscopy for neuropeptides. Journal of Histochemistry and Cytochemistry 40, 1741- 9.
Macville, M. V. E., Van Dorp, A. G. M., Wiesmeijer, K. C., Dirks, R. W., Fransen, J. A.M. & Raap, A. K. (1995) Monitoring morphology and signal during non-radioactive in situ hybridization procedures by reflection-contrast microscopy and transmission electron microscopy. Journal of Histochemistry and Cytochemistry 43, 665- 74.
Melan, M.A. & Sluder, G. (1992) Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Journal of Cell Science 101, 731- 43.
Moench, T. R., Gendelman, H. E., Clements, J. E., Narayan, O. & Griffin, D. E. (1985) Efficiency of in situ hybridization as a function of probe size and fixation technique. Journal of Virological Methods 11, 119- 30.
Ralston, E. & Hall, Z. W. (1992) Restricted distribution of mRNA produced from a single nucleus in hybrid myotubes. Journal of Cell Biology 119, 1063- 8.
Ralston, E., McLaren, R. S. & Horowitz, J. A. (1997) TfR mRNA localization to a subcompartment of the RER in skeletal myotubes. Experimental Cell Research 236, 453- 62.
Sundell, C. L. & Singer, R. H. (1991) Requirement of microfilaments in sorting of actin messenger RNA. Science 253, 1275- 7.
Urieli-Shoval, S., Meek, R. L., Hanson, R. H., Ferguson, M., Gordon, D. & Benditt, E. P. (1992) Preservation of RNA for in situ hybridization: Carnoy′s versus formaldehyde fixation. Journal of Histochemistry and Cytochemistry 40, 1879- 85.
Yaffe, D. & Saxel, O. (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270, 725- 7.
Yamawaki, M., Zurbriggen, A., Richard, A. & Vandevelde, M. (1993) Saponin treatment for in situ hybridization maintains good morphological preservation. Journal of Histochemistry and Cytochemistry 41, 105- 9.
Young, W. S. III (1993) In situ hybridization with oligodeoxyribonucleotide probes. In: In Situ Hybridization, A Practical Approach (edited by Wilkinson, D. G.), pp. 33- 44. Oxford: IRL Press at Oxford University Press.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lu, Z., Winters, C.A. & Ralston, E. Altered subcellular localization patterns of ferritin and β-actin mRNAs in muscle cultures, resulting from incomplete penetration of digoxigenin-labelled riboprobes. J Neurocytol 27, 411–418 (1998). https://doi.org/10.1023/A:1006932426837
Issue Date:
DOI: https://doi.org/10.1023/A:1006932426837