Laser Microdissection and RNA Analysis
Microdissection techniques have become an important tool to link histomorphology and pathophysiological events using modern methods of molecular biology. They allow isolation of cell clusters or even single cells precisely under optical control from complex tissue structures for further analysis of DNA, RNA, and proteins. In particular, the fragile RNA molecules can be preserved during microdissection so that gene expression and regulation measurement become feasible in a cell type-specific manner within complex tissues. This report focuses on and outlines the procedures for RNA investigation, from tissue fixation, sectioning, and staining to downstream applications (RT-PCR, mRNA quantification, and mRNA preamplification). Standards for the preparation of RNA from frozen and formalin-fixed tissues are presented. Specific protocols are given for both the isolation of RNA from small numbers of cells (50 cells) as well as for larger cell numbers. While most of the procedures are identical for the microdissection systems, special features of each technique are mentioned.
Key WordsLaser microdissection gene expression formalin fixation immunohistochemistry immunofluorescence mRNA quantification real-time PCR RNA preamplification LCM
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