Molecular Biotechnology

, Volume 10, Issue 2, pp 107–113 | Cite as

Quantitative aspects of anin situ hybridization procedure for detecting mRNAs in cells using 96-well microplates

  • Hala Zreiqat
  • Ranita Sungaran
  • C. Rolfe Howlett
  • Boban Markovic


The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitativein situ hybridization (ISH) using either image analysis or fluorescencein situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by thep-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.

Index entries

ELISA in situ hybridization mRNA microplates quantitation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Anderson, M. L. M. and Young, B. D. (1985)Nucleic Acids Hybridization. A Practical Approach (Hames, B. D. and Higgins, S. J., eds.), IRL Press, Washington, DC, pp. 73–111.Google Scholar
  2. 2.
    Stolz, L. E. and Tuan, R. S. (1996) Hybridization of biotinylated oligo(dT) for eukaryotic mRNA quantitation.Mol. Biotechnol. 6, 225–230.PubMedCrossRefGoogle Scholar
  3. 3.
    Lo, C. W. (1986) Localization of low abundance DNA sequences in tissue sections byin situ hybridization.J. Cell Sci. 81, 143–162.PubMedGoogle Scholar
  4. 4.
    Lewis, F. A., Griffiths, S., Dunnicliff, R., Wells, M., Dudding, N., and Bird, C. C. (1987) Sensitivein situ hybridization technique using biotin-streptavidinpolyalkaline phosphatase complex.J. Clin. Pathol. 40, 163–166.PubMedCrossRefGoogle Scholar
  5. 5.
    Markovic, B., Kwan, Y.-L., Nicholls, E. M., Walsh, C., and Crouch, R. L. (1992) A sensitive method for the detection of poly-A tails of mRNA using a biotinlabelled heteropolymer of dT:rA.J. Pathol. 167, 369–373.PubMedCrossRefGoogle Scholar
  6. 6.
    Britten, R. J. and Davidson, E. H. (1985)Nucleic Acids Hybridization: A Practical Approach (Hames, B. D. and Higgins, S. J., eds.), IRL, pp. 3–15.Google Scholar
  7. 7.
    Markovic, B., Wu, Z. H., Chesterman, C. N., and Chong, B. H. (1994) Quantitation of FcγRII mRNA in platelets and megakaryoblastic cell lines by a new method ofin situ hybridization.J. Immunol. Methods 172, 105–114.PubMedCrossRefGoogle Scholar
  8. 8.
    Zreiqat, H., Markovic, B., Walsh, W. R., and Howlett, C. R. (1996) A novel technique for the quantitative detection of mRNAs expression in human bone derived cells cultured on biomaterials.J. Biomater. 33, 217–223.CrossRefGoogle Scholar
  9. 9.
    Pringle, J. H., Homer, C. E., Warford, A., Kendall, C. H., and Lauder, I. (1987)In situ hybridization: alkaline phosphatase visualization of biotinylated probes in cryostat and paraffin sections.Histochem. J. 19, 488–496.PubMedCrossRefGoogle Scholar
  10. 10.
    Lawrence, J. B. and Singer, R. H. (1985) Quantitative analysis ofin situ hybridization methods for the detection of actin gene expression.Nucleic Acids Res. 13, 1777–1799.PubMedCrossRefGoogle Scholar
  11. 11.
    Hofler, H. (1990)In Situ Hybridization: Principles and Practice, (Polak, J. M. and McGee, J. O'D., eds.), Oxford University Press, Oxford, UK, chap. 2.Google Scholar
  12. 12.
    Markovic, B., Wu, Z. H., Chesterman, C. N., and Chong, B. H. (1995) Quantitation of soluble and membrane bound FcγRIIA (CD32A) mRNA in platelets and megakaryoblastic cell line (MEG-01).Br. J. Haematol. 91, 37–42.PubMedGoogle Scholar
  13. 13.
    Wu, Z. H., Markovic, B., Chesterman, C. N., and Chong, B. H. (1996) Characterisation of IgG Fe receptors on the CD34 antigen expressing cell lines (KG1 and KG-1A).Immunol. Cell Biol. 74, 57–64.PubMedCrossRefGoogle Scholar
  14. 14.
    Markovic, B., Malich, G. and Winder, C. (1995)Alternative Methods in Toxicology and the Life Sciences, vol. 11 (Goldberg, A. M. and van Zupten, L. F. M., eds.), Mary Ann Liebert, New York, pp. 283–289.Google Scholar
  15. 15.
    Gato, M., Akai, K., Murakami, A., Hashimoto, C., Tsuda, E., Ueda, M., Kawanishi, G., Takahashi, N., Ishimoto, A., Chiba, H., and Sasaki, R. (1988) Production of recombinant human erythropoietin in mammalian cells: host-cell dependency of the biological activity of the clone glycoprotein.Bio/Tech. 6, 67–71.CrossRefGoogle Scholar
  16. 16.
    Forster, A. C., McInnes, J. L., Skingle, D. C., and Symons, R. H. (1985) Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.Nucleic Acids Res. 13, 745–761.PubMedCrossRefGoogle Scholar
  17. 17.
    McInnes, J. L. and Symons, R. H. (1989)Nucleic Acid Probes (Symons, R. H., ed.), CRC Press, Boca Raton, FL, chap. 2.Google Scholar
  18. 18.
    Gato, M., Murakami, A., Akai, K., Kawanishi, G., Ueda, M., Chiba, H., and Sasaki, R. (1989) Characterization and use of monoclonal antibodies directed against human erythropoietin that recognize different antigenic determinants.Blood 74, 1415–1423.Google Scholar
  19. 19.
    Sakata, S., Enoki, Y., Shimizu, S., Hattori, M., and Ueda, M. (1995) Correlation between a sandwich ELISA and an in-vitro bioassay for erythropoietin in human plasma.Br. J. Haematol. 91, 562–565.PubMedGoogle Scholar
  20. 20.
    Taxman, D. J., Lee, E. S., and Wojchowski, D. M. (1993) Receptor-targeted transfection using stable maleimido-transferrin/thio-poly-lysine conjugates.Anal. Biochem. 213, 97–103.PubMedCrossRefGoogle Scholar
  21. 21.
    Alard, P., Lantz, O., Sebagh, M., Calvo, C. F., Weill, D., Chavanel, G., Senik, A., and Charpentier, B. (1993) A versatile ELISA-PCR assay for mRNA quantitation from a few cells.BioTechniques 15, 730–737.PubMedGoogle Scholar
  22. 22.
    Ambinder, R. F., Charache, P., Staal, S., Wright, P., Forman, M., Hayward, S. D. and Hayward, G. S. (1986) The vector homology problem in clinical nucleic acid hybridization of clinical specimens.J. Clin. Microbiol. 24, 16–20.PubMedGoogle Scholar

Copyright information

© Humana Press Inc 1998

Authors and Affiliations

  • Hala Zreiqat
    • 1
  • Ranita Sungaran
    • 2
  • C. Rolfe Howlett
    • 1
  • Boban Markovic
    • 3
  1. 1.Bone Biomaterials UnitThe University of New South WalesSydneyAustralia
  2. 2.Centre for Thrombosis and Vascular Research, School of PathologyThe University of New South WalesSydneyAustralia
  3. 3.Chemical Safety and Applied Toxicology Unit, Department of Safety ScienceThe University of New South WalesSydneyAustralia

Personalised recommendations