Abstract
In situ hybridization to messenger RNA (mRNA) in complex tissues, such as the brain, allows the localization of gene expression to functionally distinct regions. It has been difficult to measure relative changes in gene expression within these regions because of the poor cellular resolution afforded by radioactively labeled probes and problems associated with densitometric analysis by counting silver grain deposition. Fluorescence in situ hybridization, using probes directly labeled with dyes that exhibit high quantum yield, provides both high-resolution localization of mRNA and high sensitivity for detection of hybridized probe. Digital image capture of fluorescence is readily amenable to densitometric analysis, thereby allowing relative quantification of mRNA expression in single cells or discrete brain nuclei. In this chapter, we describe protocols suitable for measuring relative changes in gene expression within individual cells of brain sections mounted on glass slides.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mrkusich, E. M., Kivell, B. M., Miller, J. H., and Day, D. J. (2004) Abundant expression of mu and delta opioid receptor mRNA and protein in the cerebellum of the fetal, neonatal and adult rat. Dev. Brain. Res. 148, 213–222.
Ueda, H., Nozaki, M., and Satoh, M. (1991) Multiple opioid receptors and GTP-binding proteins Comp. Biochem. Physiol. 98, 157–169.
Knapp, R. J., Malatynska, E., Collins, N., et al. (1995) Molecular biology and pharmacology of cloned opioid receptors. FASEB J. 9, 516–525.
Zastawny, R. L., George, S. R., Nguyen, T., et al. (1994) Cloning, characterization, and distribution of a mu-opioid receptor in rat brain. J. Neurochem. 62, 2099–2105.
Abood, M. E., Noel, M. A., Farnsworth, J. S., and Tao, Q. (1994) Molecular cloning and expression of a delta-opioid receptor from rat brain. J. Neurosci. Res. 37, 714–719.
Minami, M., Toya, T., Katao, Y., et al. Cloning and expression of a cDNA for the rat kappa-opioid receptor. FEBS Lett. 329, 291–295.
Wilcox, J. N. (1993) Fundamental principles of in situ hybridization. J. Histochem. Cytochem. 41, 1725–1733.
Henderson, Z. (1996) In Situ Hybridization Techniques for the Brain, IBRO Handbook series: Methods in the Neurosciences, volume 17, John Wiley and Sons, New York.
Rychlik, W. and Rhoads, R. E. (1989) A computer program for choosing the optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res. 17, 8543–8551.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2 ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Lab FAQs. Roche Applied Science. http://www.roche-applied-science.com/labfaqs.
Milligan, J. F., Groebe, D. R., Witherell, G.W., and Uhlenbeck, O. C. (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798.
Nucleic Acid Isolation and Purification Manual. (2002) Roche Applied Science.
Nonradioactive In Situ Hybridization Application Manual. (2002) Roche Applied Science.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Day, D.J., Mrkusich, E.M., Miller, J.H. (2007). Comparative Quantitation of mRNA Expression in the Central Nervous System Using Fluorescence In Situ Hybridization. In: Hilario, E., Mackay, J. (eds) Protocols for Nucleic Acid Analysis by Nonradioactive Probes. Methods in Molecular Biology, vol 353. Humana Press. https://doi.org/10.1385/1-59745-229-7:125
Download citation
DOI: https://doi.org/10.1385/1-59745-229-7:125
Publisher Name: Humana Press
Print ISBN: 978-1-58829-430-2
Online ISBN: 978-1-59745-229-8
eBook Packages: Springer Protocols