4 June 2002 Importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics
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Proceedings Volume 4625, Clinical Diagnostic Systems: Technologies and Instrumentation; (2002) https://doi.org/10.1117/12.469773
Event: International Symposium on Biomedical Optics, 2002, San Jose, CA, United States
Abstract
Gene expression microarray analyses of mixtures of cells approximate a weighted average of the gene expression profiles (GEPs) of each cell type according to its relative abundance in the overall cell sample being analyzed. If the targeted subpopulation of cells is in the minority, or the expected perturbations are marginal, then such changes will be masked by the GEP of the normal/unaffected cells. We show that the GEP of a minor cell subpopulation is often lost when that cell subpopulation is of a frequency less than 30 percent. The GEP is almost always masked by the other cell subpopulations when that frequency drops to 10 percent or less. Several methodologies can be employed to enrich the target cells submitted for microarray analyses. These include magnetic sorting and laser capture microdissection. However, high-throughput flow cytometry/cell sorting overcomes many restrictions of experimental enrichment conditions. This technology can also be used to sort smaller numbers of cells of specific cell subpopulations and subsequently amplify their mRNAs before microarray analyses. When purification techniques are applied to unfixed samples, the potential for changes in gene levels during the process of collection is an additional concern. High-throughput cell separation technologies are needed that can process the necessary number of cells expeditiously in order to avoid such uncontrolled changes in the target cells GEP. In cases where even the use of HTS yields only a small number of cells, the mRNAs (after reverse transcription to cDNA's) must be amplified to yield enough material for conventional microarray analyses. However, the problem of using microamplification PCR methods to expand the amount of cDNAs (from mRNAs) is that it is very difficult to amplify equally all of the mRNAs. Unequal amplification leads to a distorted gene expression profile on the microarray. Linear amplifications is difficult to achieve. Unfortunately, present-day gene-chips need to be about 100 times more sensitive than they are now to be able to do many biologically and biomedically meaningful experiments and clinical tests.
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James F. Leary, Peter Szaniszlo, Tarl W. Prow, Lisa M. Reece, Nan Wang, David M. Asmuth, "Importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics", Proc. SPIE 4625, Clinical Diagnostic Systems: Technologies and Instrumentation, (4 June 2002); doi: 10.1117/12.469773; https://doi.org/10.1117/12.469773
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