Expression profiling from microdissected samples
DESCRIPTION (provided by applicant): One objective of the proposal is to develop a product line that will consist of amplified RNA (aRNA) derived from pure populations of cells from various regions of mouse brain. The target cells will be selected by Laser Capture Micro-dissection (LCM). The product line will include aRNA derived from normal mice and from mutant mice that serve as models of human neurodegenative disorders, including Alzheimer's disease, Huntington's disease, and ataxia telangiectasia. The product will be targeted to researchers carrying out expression profiling studies that aim to understand the molecular basis of normal neurological function and the molecular pathology of neurological disease. During the first year, efforts will be focused on scaling up procedures for sample processing, micro-dissection, and RNA isolation and amplification from normal mice. During the second year the procedures will be applied to the mutant strains. The aRNA will be produced using T7-mediated in vitro transcription. In order to help meet the anticipated Production-scale goals, efforts will be directed to adapting the procedures, especially RNA amplification, to a robotic platform. In order to assess the RNA from micro-dissected samples for quality assurance purposes, molecular markers will be identified to use for verifying that the RNA is derived from the intended cell population. Efforts will also be directed to improving methods for identifying target cells for micro-dissection in samples such as tumors, where target cells (e.g. malignant cells) cannot always be distinguished by histological staining. The methods will be compatible with isolation of intact RNA from the selected cells, so that it can be amplified for use in microarray expression profiling assays. To meet this goal we will develop rapid fluorescent detection methods using primary fluorescent antibodies directed to tumor antigens, and we will also adapt detection of target cells in transgenic animals expressing Green Fluorescent Protein for use with LCM. Achieving these goals will provide aRNA from distinct cellular subtypes to neuroscience researchers who do not currently have access to this resource. Use of aRNA from defined cell subtypes, as opposed to bulk tissue, will improve the ability to make biologically meaningful conclusions from expression profiling experiments carried out on highly heterogeneous tissues such as brain. Another goal of the project is to release a kit for recovery of high-quality total RNA from microdissected samples, especially those obtained by LCM. The kit will be of general use to life science researchers working with micro-scale samples.
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