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J Vis Exp. 2015 Apr 11;(98). doi: 10.3791/52510.

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling.

Author information

1
Department of Psychiatry and Neurosciences, Faculty of Medicine, Université Laval; Centre de recherche de l'Institut universitaire en santé mentale de Québec.
2
Department of Psychiatry and Neurosciences, Faculty of Medicine, Université Laval; Centre de recherche de l'Institut universitaire en santé mentale de Québec; martin.levesque@crulrg.ulaval.ca.

Abstract

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

PMID:
25939046
PMCID:
PMC4541564
DOI:
10.3791/52510
[Indexed for MEDLINE]
Free PMC Article

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