Abstract
Laser-capture microdissection (LCM) allows for retrieval of specific cell populations in situ. By combining immunofluorescent labeling with LCM, mRNAs can be probed by qRT-PCR for determining in situ gene expression during health and disease. This approach permits obtaining and analyzing histologically enriched cell populations in a tissue that can be hardly obtained from other methods such as white matter astrocytes from rodents or any individual cell population from archival human or rodent brain tissues. Herein, we present our methodology of laser-captured mouse spinal cord white matter astrocytes, which can be adapted for any cell type in CNS tissue and low RNAse containing tissues. The methods presented with an emphasis on tips and advices include the cryostat section preparation from snap-frozen tissue, an adapted immunofluorescent labeling, a brief overview of LCM using a UV-based technology with polyethylene membrane glass slides, procedures for direct use of RNA from lysis buffer vs. column-based purified RNA, RNA quality/quantity assessment, the reverse transcription and preamplification steps used before real-time qPCR analysis.
Similar content being viewed by others
References
Böhm C, Newrzella D, Sorgenfrei O (2005) Laser microdissection in CNS research. Drug Discov Today 10:1167–1174
Espina V, Milia J, Wu G, Cowherd S, Liotta LA (2006) Laser capture microdissection. Methods Mol Biol 319:213–229
Murugesan N, Macdonald J, Ge S, Pachter JS (2011) Probing the CNS microvascularendothelium by immune-guided laser-capture microdissection coupled to quantitative RT-PCR. Methods Mol Biol 755:385–394
Cahoy JD, Emery B, Kaushal A et al (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 28:264–278
Shaw KJ, Thain L, Docker PT, Dyer CE et al (2009) The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths. Anal Chim Acta 652:231–233
Guillot F, Garcia A, Salou M et al (2015) Transcript analysis of laser capture microdissected white matter astrocytes and higher phenol sulfotransferase 1A1 expression during autoimmune neuroinflammation. J Neuroinflammation 12:130
Demarest TG, Murugesan N, Shrestha B, Pachter JS (2012) Rapid expression profiling of brain microvascular endothelial cells by immuno-laser capture microdissection coupled to TaqMan(®) low density array. J Neurosci Methods 206:200–204
Ellem KA, Colter JS (1961) A consideration of the ribonucleic acid depolymerase-inhibitor systems of mouse tissues. J Cell Comp Physiol 58:267–276
Ordway GA, Szebeni A, Duffourc MM et al (2009) Gene expression analyses of neurons, astrocytes, and oligodendrocytes isolated by laser capture microdissection from human brain: detrimental effects of laboratory humidity. J Neurosci Res 87:2430–2438
Brown AL, Smith DW (2009) Improved RNA preservation for immunolabeling and laser microdissection. RNA 15:2364–2374
Acknowledgments
We are grateful to Francoise Gros and the Genomics platform of Nantes (Biogenouest Genomics) core facility for the technical support with the Agilent LabChips. Supported by Region Pays de la Loire, Rotary/Fondation de la Recherche sur le Cerveau (FRC), and INSERM.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Nicot, A.B., Rambeau, J., Guillot, F., Garcia, A., Laplaud, D.A. (2018). Immuno-Guided Laser-Capture Microdissection of Glial Cells for mRNA Analysis. In: Murray, G. (eds) Laser Capture Microdissection. Methods in Molecular Biology, vol 1723. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7558-7_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7558-7_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7557-0
Online ISBN: 978-1-4939-7558-7
eBook Packages: Springer Protocols