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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZATION AND ENHANCEMENT OF IMMUNE RESPONSES OF CALVES Title: Laser Capture Microdissection Revisited as a Tool for Transcriptomic Analysis: Application of an Excel-Based qPCR Preparation Software (PREXCEL-Q)

Authors
item Sow, Fatoumata - IOWA STATE UNIVERSITY
item Gallup, Jack - IOWA STATE UNIVERSITY
item Sacco, Randy
item Ackermann, Mark - IOWA STATE UNIVERSITY

Submitted to: International Journal of Biomedical Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 23, 2009
Publication Date: June 20, 2009
Citation: Sow, F.B., Gallup, J.M., Sacco, R.E., Ackermann, M.R. 2009. Laser Capture Microdissection Revisited as a Tool for Transcriptomic Analysis: Application of an Excel-Based qPCR Preparation Software (PREXCEL-Q). International Journal of Biomedical Science. 5(2):105-124.

Interpretive Summary: A refinement of a technique is described for the isolation of white blood cells from tissues using direct visualization with a microscope and laser capture. We describe a series of protocols for its use and for use in additional experiments. The approximate time needed to complete these experiments is 12.5 to 17.5 hours. This technology will allow scientists to study single cells isolated from control or infected tissues.

Technical Abstract: The ability to reliably analyze cellular and molecular profiles of normal or diseased tissues is frequently obfuscated by the inherent heterogeneous nature of tissues. Laser Capture Microdissection (LCM) is an innovative technique that allows the isolation and enrichment of pure subpopulations of cells from tissues under direct microscopic visualization. Material obtained from captured cells can be used for downstream assays, including gene expression studies such as microarrays, proteomic assays such as western blotting, cDNA library generation, and DNA genotyping. We describe a series of LCM protocols coupled to PREXCEL-Q-based qPCR for examining macrophage populations. We focus on two LCM approaches: “laser cutting” and “laser capture” for which the calculations for reagents and time involved have been pre-determined for 10 samples. One can expect the entire procedure for laser cutting coupled to qPCR to take approximately 12.5-15 h, and laser capture coupled to qPCR to take approximately 13.5-17.5 h.

Last Modified: 12/21/2014
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