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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    ABSTRACT: MicroRNAs play important roles in laryngeal carcinoma and other cancers. However, the expression of microRNAs in paracancerous tissue has been studied less. Here, using laser capture microdissection (LCM), we detected the expression of microRNAs in paracancerous tissues. Among all down-regulated microRNAs in the center area of tumor tissues, only miR-30b expression was significantly reduced in paracancerous tissues compared to surgical margins. Therefore, to further investigate the effect of miR-30b on laryngeal carcinoma, we stably overexpressed miR-30b in laryngeal carcinoma cell line HEp-2 cells. It was found that although there was no significant difference in cell viability between miR-30b overexpressed cells and control HEp-2 cells, p53 expression was obviously enhanced in miR-30b overexpressed cells. Whether miR-30b could improve the anti-tumor effect of adenovirus-p53 (Ad-p53) in laryngeal carcinoma and other cancer cell lines was also evaluated. It was found that in miR-30b overexpressed HEp-2 cells, p53-mediated tumor cell apoptosis was obviously increased both in vitro and in vivo. MDM2-p53 interaction might be involved in miR-30b-mediated anti-tumor effect. Together, results suggested that miR-30b could modulate p53 pathway and enhance p53 gene therapy-induced apoptosis in laryngeal carcinoma, which could provide a novel microRNA target in tumor therapy.
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    ABSTRACT: Laser capture microdissection (LCM) is a powerful histology-based technique for isolation of pure cells from their heterogenous environments as well as from cytological preparations and live cells. The LCM technique allows differentiation between normal and morphologically abnormal cells as distinct cell populations from the heterog-enous mixture, and it is possible to investigate subcellular profiles with great accuracy. Biomolecules such as RNA, DNA, and protein extracted from microdissected cells can be used for variety of downstream applications, including polymerase chain reaction gene expression studies, microarray analysis, and proteomic analysis. The success of LCM in extraction of DNA, RNA, and protein from a small number of cells suitable for downstream applications depends on several critical factors, such as proper tissue collection and fixation techniques, staining procedures, nucleic acid or protein extraction methods, and storage. Here we discuss and describe protocols from our laboratory that are optimized for LCM of endometrial immune cells, endothelial cells, and trophoblasts. Abbreviations aRNA antisense RNA cDNA complimentary DNA IR infra-red LCM laser capture microdissection LPC laser pressure catapult OCT optimal cutting temperature PEN polyethylene naphthalate UV ultarvoilet uNK uterine natural killer cells IntroductIon Laser capture microdissection (LCM) is a powerful method for isolating pure, homogenous cell popula-tions from specific microscopic regions of complex tissue sections. This technique has greatly contrib-uted to the exponential growth in gene expression analysis, proteomics, and biomarker discovery. LCM technology was originally developed by Emmert-Buck and colleagues at the National Institutes of Health (Bethesda, MD, USA) for accurate and efficient dissec-tion of cells from histological tissue sections of solid tumors. 1,2 The technology was rapidly commercial-ized in 1997 through a collaborative research and development agreement partnership with Arcturus Engineering (Sunnyvale, CA, USA). The principle of this technique relied on using a low-power infrared laser to melt a special thermoplastic film (attached to the cap) over the cells or tissue areas of interest. The cells of interest, firmly attached on the thermoplastic polymer of the cap, will be sheared away when the cap is removed from the heterogenous tissue section. This technique ensured exact cellular morphology and biomolecular integrity (RNA, DNA, and protein) of the captured cells. Arcturus Engineering success-fully commercialized PixCell II, PixCell IIe, and the fully automated Veritas LCM equipment over the years. LCM remains the standard terminology for a vari-ety of instruments. In general, regardless of the type of laser used, there are three classes of LCM systems based on the type of laser used and collection of dis-sected material: infrared (IR) LCM, 1,2 ultraviolet (UV)
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    ABSTRACT: To identify proteins and (molecular/biological) pathways associated with differences between benign and malignant epithelial ovarian tumors.
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