Quantitative aspects of anin situ hybridization procedure for detecting mRNAs in cells using 96-well microplates
- 64 Downloads
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 entriesELISA in situ hybridization mRNA microplates quantitation
Unable to display preview. Download preview PDF.
- 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
- 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
- 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
- 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.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
- 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.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