Abstract
This chapter describes the procedure of in situ hybridization-guided laser-capture microdissection performed on postmortem human brain tissue. This procedure permits the precise collection of brain tissue within anatomically defined brain nuclei that is enriched with mRNA. The chapter emphasizes the specific handling of postmortem tissue and preservation of RNA integrity to ensure high-quality gene profiling. Downstream procedures including mRNA amplification, gene profiling using high-density microarray chips, and confirmation with quantitative real-time polymerase chain reaction (qPCR) are described. PCR primer design and cDNA quantification required for qPCR are delineated.
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
Similar content being viewed by others
References
Bernard, R., Kerman IA, Meng F, Evans SJ, Amrein I, Jones EG, Bunney WE, Akil H, Watson SJ, Thompson RC (2009) Gene expression profiling of neurochemically-defined regions of the human brain by in situ hybridization-guided laser capture microdissection. J Neurosci Methods, 178:46–54.
Bernard, R., Kerman IA, Thompson RC, Jones EG, Bunney WE, Barchas JD, Schatzberg AF, Myers RM, Akil H, Watson SJ (2011) Altered expression of glutamate signaling, growth factor, and glia genes in the locus coeruleus of patients with major depression. Mol Psychiatry, 16: in press.
Atz, M., Walsh D, Cartagena P, Li J, Evans S, Choudary P, Overman K, Stein R, Tomita H, Potkin S, Myers R, Watson SJ, Jones EG, Akil H, Bunney WE Jr, Vawter MP (2007) Methodological considerations for gene expression profiling of human brain. J Neurosci Methods, 163:295–309.
Li, J.Z., Vawter MP, Walsh DM, Tomita H, Evans SJ, Choudary PV, Lopez JF, Avelar A, Shokoohi V, Chung T, Mesarwi O, Jones EG, Watson SJ, Akil H, Bunney WE Jr, Myers RM (2004) Systematic changes in gene expression in postmortem human brains associated with tissue pH and terminal medical conditions. Hum Mol Genet, 13:609–16.
Tomita, H. Vawter MP, Walsh DM, Evans SJ, Choudary PV, Li J, Overman KM, Atz ME, Myers RM, Jones EG, Watson SJ, Akil H, Bunney WE (2004) Effect of agonal and postmortem factors on gene expression profile: quality control in microarray analyses of postmortem human brain. Biol Psychiatry, 55:346–52.
Rehncrona, S., I. Rosén, B.K. Siesjö. (1981) Brain lactic acidosis and ischemic cell damage: 1. Biochemistry and neurophysiology. J Cereb Blood Flow Metab, 1:297–311.
Yates, C.M. Butterworth J, Tennant MC, Gordon A. (1990) Enzyme activities in relation to pH and lactate in postmortem brain in Alzheimer-type and other dementias. J Neurochem, 55:1624–30.
Schoor, O. Weinschenk T, Hennenlotter J, Corvin S, Stenzl A, Rammensee HG, Stevanović S. (2003) Moderate degradation does not preclude microarray analysis of small amounts of RNA. Biotechniques, 35:1192–6, 1198–201.
Greene, J.G., R. Dingledine, J.T. Greenamyre, (2005) Gene expression profiling of rat midbrain dopamine neurons: implications for selective vulnerability in parkinsonism. Neurobiol Dis, 18:19–31.
Torres-Munoz, J.E. Van Waveren C, Keegan MG, Bookman RJ, Petito CK. (2004) Gene expression profiles in microdissected neurons from human hippocampal subregions. Brain Res Mol Brain Res, 127:105–14.
Neal, C.R., Jr., H. Akil, S.J. Watson, Jr., (2001) Expression of orphanin FQ and the opioid receptor-like (ORL1) receptor in the developing human and rat brain. J Chem Neuroanat, 22:219–49.
Kerman, I.A. Buck BJ, Evans SJ, Akil H, Watson SJ. (2006) Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression. J Neurosci Methods, 153:71–85.
Rozen, S. Skaletsky H, (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol, 132:365–86.
Zuker, M., (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res, 31:3406–15.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Bernard, R., Burke, S., Kerman, I.A. (2011). Region-Specific In Situ Hybridization-Guided Laser-Capture Microdissection on Postmortem Human Brain Tissue Coupled with Gene Expression Quantification. In: Murray, G. (eds) Laser Capture Microdissection. Methods in Molecular Biology, vol 755. Humana Press. https://doi.org/10.1007/978-1-61779-163-5_29
Download citation
DOI: https://doi.org/10.1007/978-1-61779-163-5_29
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-162-8
Online ISBN: 978-1-61779-163-5
eBook Packages: Springer Protocols