Laser microdissection: sample preparation and applications

Abstract

Laser microdissection (LM) is a rapid, easy, one-step, and efficient method of conserving and isolating single cell or cell clusters from fixed tissue sections under direct microscopic visualization. LM is currently the only method that can be used to isolate homogeneous cells from heterogeneous tissue. This method was first developed for tumor analysis. Multiple generations of LM instruments are available on the market. For instance, the Veritas™ microdissection combines the LM system based on the IR with UV laser cutting. The desired cells can be harvested specific and wanted cells can be harvested directly by cutting target cells abroad from unwanted cells. The extracted cells are then utilized in various disciplines of experimental and clinical biology, such as genomic analysis and proteomics. LM is a an efficient and powerful tool that facilitates the analysis of small amounts of molecules isolated from a complex tissue, thus offering new opportunities to understand physiological and fundamental processes. Here, we describe a method of preparing paraffin sections of maize roots for laser microdissection to three parts (stele, cortex and outer layers) by LM using a microwave embedding method. This approach allows RNA to be extracted from each type of tissue separately. The RNA integrity of our samples following LM ranged between 7 and 7.2, indicating that this RNA can be reliably used for further analysis. The work reported in this paper highlights how advances in protocols and methods have made LM a powerful and sometimes essential tool for genomic and proteomic analyses of tiny amounts of biomolecules extracted from a few cells isolated from a complex tissue in their physiological context, thus offering a new pathway to understand fundamental, physiological and/or patho-physiological processes.