|Morimoto, Motoko - USUHS, BETHESDA, MD|
|Morimoto, Masahiro - "|
|Whitemire, Jeannette - "|
|Star, Robert - "|
|Gause, William - "|
Submitted to: In: PCR Primer: A Laboratory Manual
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 15, 2003
Publication Date: December 1, 2003
Citation: Morimoto, M., Morimoto, M., Whitemire, J., Star, R., Urban Jr, J.F., Gause, W. 2003. In situ localization of gene expression using laser capture micro dissection. In: PCR Primer: A Laboratory Manual. 11:135-148. Interpretive Summary: A powerful new technique called laser capture micro dissection (LCM), that was originally developed to isolate individual cells from tissues derived from patients with cancer, has now been broadened for use in tissues invaded by parasites. This provides a very sensitive procedure for asking questions about molecular event within the affected tissue, and can provide insight into the mechanisms that control infection. Both gene and protein expression at the site can be measured. There is a step by step description of the procedures need to obtain the tissue and prepare it for LCM, followed by the intricacies required to successfully isolate products for the measurement of gene and protein expression. The results demonstrate that local responses to infection evolve more rapidly and with the attraction of specific cells and their products than can not be detected at other sites in the body of the infected host. The information obtained from these studies will help devise procedures to more efficiently control infection and limit the tissue damage that often accompanies the host response. It is also possible to change the diet of the host and assess the local changes in the host response as a result of dietary modifications. This information will be of general benefit to those scientists interested in the role of nutrition in control of immunity to infectious diseases.
Technical Abstract: The lymph nodes of the immune system are complex organs with specific microenvironments in which lymphoid lineages differentiate and become committed to specific effector functions. Elucidation of the proteins and genes expressed by lymphocytes in situ can potentially provide important information regarding their function in their natural milieu. The technique of laser capture micro dissection (LCM), which was originally developed to study gene expression in tumor cells, is well-suited for examination of cell populations in lymphoid organs or other heterogeneous tissue microenvironments. Other techniques that examine cell function in situ include immunohistology and in situ hybridization. These techniques are frequently not sufficiently sensitive to detect physiological up regulation of proteins or specific mRNA expression, and they usually cannot be used to detect more than one or two expressed molecules at a time. Various micro dissection techniques have been used to remove single cells from tissue sections for further analysis. For example, micropipettes have been successfully used to remove single cells from germinal centers of B cell follicles in the lymph node to study B cell differentiation. However, these techniques are laborious and difficult to perform and thus are not practical in many laboratories. The development of LCM provides a rapid and readily learned technique for removal of specific cells or tissue regions from a tissue section on a microscope slide. RNA or protein can be purified and used in a variety of assays in the laboratory including real-time reverse transcriptase (RT)-PCR, allowing the analysis of the expression of a number of different genes from the same captured sample.