Article

Laser-capture microdissection

Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Blvd. MS 4E3, Manassas, Virginia, USA.
Nature Protocol (Impact Factor: 8.36). 02/2006; 1(2):586-603. DOI: 10.1038/nprot.2006.85
Source: PubMed

ABSTRACT Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1-1.5 h.

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    • "The quantity of RNA obtained after LCM is typically in the order of several picograms to a few nanograms, depending on the amount and type of cells captured. A quantity of 10 pg of total RNA per cell is commonly quoted, (e.g., Espina et al., 2006), and "
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    • "Some multicellular organisms , such as Caenorhabditis elegans (Sulston et al. 1983) and zebra fish (Aanes et al. 2011, 2014), have known numbers of cells at defined developmental stages. Laser capture microdissection enables the number of cells used for RNA extraction to be determined when working with solid tissues (Espina et al. 2006). In such cases, heterologous RNA (e.g., External RNA Fig. 4 Distribution of gene dosage responses in G. dolichocarpa. "
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    • "The importance to evaluate biological mechanisms on single cell resolution is most evident, especially with regard to the analysis of human disease-relevant interfaces, such as clinical specimens. Laser microdissection with subsequent microarray analysis and reverse transcriptase-quantitative PCR (RT-qPCR) is the most suitable investigative tool for gene expression analysis in basic and clinical research (Cohen et al., 2002; Espina et al., 2006; Lotz et al., 2006). Nevertheless, limited RNA yields and qualities of laser microdissected tissue make gene expression analysis unreliable. "
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