Laser microdissection and gene expression analysis on formaldehyde-fixed archival tissue
Clemens D. CohenHermann-Josef GröneElisabeth GrönePeter J. NelsonDetlef SchlöndorffMatthias Kretzler
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Laser capture microdissection
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Histologic and cytologic changes are central to the diagnosis and classification of many disease processes, particularly neoplasms. The correlation of these changes with genomics, proteomics, and molecular pathways entails refined microdissection techniques that are frequently used to procure a pure population of cells from complex tissue. Here we review the past, present, and future of some of these new advances in microdissection techniques including manual techniques, laser microdissection, laser capture microdissection, and laser catapulting.
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To quantify gene expression in tumor cells from human head and neck squamous cell carcinomas (HNSCC) using laser capture microdissection (LCM).Histopathologically identified HNSCC cells were microdissected from frozen sections, RNA was isolated, and vascular endothelial growth factor (VEGF) gene expression was measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR).Two human HNSCC tumor samples and matched normal mucosal biopsies and five human xenograft tumor specimens were harvested, embedded, and frozen in OCT. The frozen tumors were sectioned to 8 to 10 mum in thickness, and hematoxylin-eosin (H&E) staining was performed before LCM. An estimated 2,000 to 3,000 tumor cells were microdissected from frozen sections and processed for RNA isolation. mRNA for VEGF was analyzed by real time RT-PCR (TaqMan) with commercially available primers and probes.Two thousand to 3000 cells were necessary to obtain a suitable quantity of RNA for subsequent gene expression study by real-time RT-PCR. The gene expression of VEGF, a major tumor angiogenic factor, was tested in microdissected HNSCC and compared with uninvolved normal mucosal controls. A greater than seven-fold increase of VEGF expression in tumor specimens versus mucosal controls was observed.LCM is a novel sample conserving technique that allows the precise selection of tumor cells from a heterogeneous architecture. The combination of LCM and real-time RT-PCR appears particularly efficacious for studying HNSCC molecular pathogenesis and identifying tissue-specific biomarkers.
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ABSTRACT Background and aim: Laser capture microdissection is used to obtain pure cell populations, thus allowing the study of molecular pathological mechanisms from specific cell populations, yet RNA from tissues fixed in formalin (the most common fixative for analysis of gene expression) is usually degraded during microdissection and RNA extraction. In this study we investigate the best fixative for use in the preparation of human tissues for laser capture microdissection, and for the preservation of RNA molecules and histomorphologic structure. Methods: Here we examine Methacarn, Carnoy's solution, 4% paraformaldehyde and 10% neutral‐buffered formalin. Forty‐three sides of various human organs were compared to determine the tissue‐fixing ability and RNA quality according to the fixatives. Results: Methacarn was the best suitable fixative for histomorphologic preservation as well as protection against RNA degradation after microdissection. However, Methacarn was not good for preservation of proteins. Conclusions: Methacarn is an excellent fixative for human tissue samples obtained by microdissection. It is an optimal fixative that is not only excellent for preservation of morphology, but also RNA extraction.
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The kidney consists of many functional modules called nephrons. Each nephron has a tubular structure made up of several structurally and functionally distinct segments. The analysis of individual segments requires the use of microdissection techniques. We describe protocols that have been used to successfully isolate messenger RNA from proximal tubules of both freshly prepared and archival samples using laser capture microdissection and laser-manipulated microdissection.
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Tissue microdissection is potentially one of the most useful techniques in molecular pathology. Laser-assisted microdissection has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with multiplex molecular approaches, it is now feasible to study genetic alterations and isolate genes and proteins in defined cell populations from complex normal and diseased tissues. The fundamental advantage of this technique is the possibility of capturing single cells from which high-quality DNA and mRNA can be isolated for analysis of sequence and quantitation of expression. Moreover, the integration of laser-assisted microdissection and proteomic analysis could identify novel protein markers for disease. The advent of laser-assisted microdissection is likely to have a profound impact on molecular pathology. Copyright © 1999 John Wiley & Sons, Ltd.
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One difficulty in studying molecular changes of tumours has been the inability to isolate DNA and RNA from a homogeneous cell population. The combination of several new technologies should help overcome these hurdles. Microdissection is a technique for rapid and easy procurement of a pure cellular subpopulation away from its complex tissue milieu. Laser-assisted microdissection has recently been identified as a quick, simple and effective method by which microdissection of complex tissue specimens can be routinely performed for molecular analysis. With the advent of laser microdissection, cDNA libraries can be developed from pure cells obtained directly from stained neoplastic tissue, and microarrays of thousands of genes can now be used to examine gene expression in microdissected tumour tissue samples. This review will concentrate on the application of different microdissection techniques in the area of cancer research.
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Laser microdissection provides a rapid isolating and purification modern method for biological tissues and cellular preparations.It directly cuts objects from heterogeneous tissues under the microscope.This review concerned an up-to-date advance in Laser microdissection techniques which are applied on to the plant cell and molecular biology researches in regard with the sample preparation,gene expression analysis,DNA and protein studies etc.Meanwhile,possible directions for future development of laser microdissection in plant research were put forward.
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