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.
SourceAvailable from: Mohamed Elzek[Show abstract] [Hide abstract]
ABSTRACT: In the past decade, there has been an increasing interest in applying proteomics to assist in understanding the pathogenesis of ovarian cancer, elucidating the mechanism of drug resistance, and in the development of biomarkers for early detection of ovarian cancer. Although ovarian cancer is a spectrum of different diseases, the strategies for diagnosis and treatment with surgery and adjuvant therapy are similar across ovarian cancer types, increasing the general applicability of discoveries made through proteomics research. While proteomic experiments face many difficulties which slow the pace of clinical applications, recent advances in proteomic technology contribute significantly to the identification of aberrant proteins and networks which can serve as targets for biomarker development and individualized therapies. This review provides a summary of the literature on proteomics' contributions to ovarian cancer research and highlights the current issues, future directions, and challenges. We propose that protein-level characterization of primary lesion in ovarian cancer can decipher the mystery of this disease, improve diagnostic tools, and lead to more effective screening programs.Cancer and metastasis reviews 03/2015; 34(1). DOI:10.1007/s10555-014-9547-8 · 6.45 Impact Factor
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ABSTRACT: Mucosal tissues are critical immune effector sites containing complex populations of leukocytes in a tissue microenvironment that remains incompletely understood. We identify and quantify in human distal colorectal tissue absolute mucosal CD3+ lymphocytes, including CD4+ and CD8+ subsets, by direct visualization using immunohistochemistry (IHC), immunofluorescence (IF), and an automated counting protocol (r2=0.90). Sigmoid and rectal mucosal tissues are both densely packed with T lymphocytes in the mucosal compartment. Both compartments had similar densities of CD3+ T lymphocytes with 37,400 ± 2,801 cells/mm3 and 33,700 ± 4,324 cell/mm3, respectively. Sigmoid mucosa contained 57% CD3+CD4+ and 40% CD3+CD8+ T lymphocytes which calculates to 21,300 ± 1,476/mm3 and 15,000 ± 275/mm3 T lymphocytes, respectively. Rectal mucosa had 57% CD3+CD4+ and 42% CD3+CD8+ or 21,577 ± 332, and 17,090 ± 1,206 cells/mm3, respectively. By comparison, sigmoid mucosal biopsies subjected to conventional collagenase digestion, mononuclear cell (MMC) isolation and staining for flow cytometry yielded 4,549 ± 381/mm3 and 2,708 ± 245/mm3 CD4+ and CD8+ T lymphocytes. These data suggest only ~20.7% recovery compared to IHC results for these markers. Further studies will determine if this reflects a selective bias in only CD3+, CD4+ and CD8+ T cells or can be generalized to all flow-analyzed cells from mucosal tissues for phenotyping and functional testing.PLoS ONE 04/2015; 10(4):e0122723. DOI:10.1371/journal.pone.0122723 · 3.53 Impact Factor
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ABSTRACT: Laser capture microdissection (LCM) facilitates the isolation of individual cells from tissue sections, and when combined with RNA amplification techniques, it is an extremely powerful tool for examining genome-wide expression profiles in specific cell-types. LCM has been widely used to address various biological questions in both animal and plant systems, however, no attempt has been made so far to transfer LCM technology to macroalgae. Macroalgae are a collection of widespread eukaryotes living in fresh and marine water. In line with the collective effort to promote molecular investigations of macroalgal biology, here we demonstrate the feasibility of using LCM and cell-specific transcriptomics to study development of the brown alga Ectocarpus siliculosus. We describe a workflow comprising cultivation and fixation of algae on glass slides, laser microdissection, and RNA amplification. To illustrate the effectiveness of the procedure, we show qPCR data and metrics obtained from cell-specific transcriptomes generated from both upright and prostrate filaments of Ectocarpus.Frontiers in Plant Science 02/2015; 6(54). DOI:10.3389/fpls.2015.00054 · 3.64 Impact Factor