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The Importance of High-Throughput Cell Separation Technologies for Genomics Proteomics-Based Clinical Diagnostics


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 wher that frequency drops to 10 percent or less. On the basis of these results one should always assume that the GEP of a given cell subpopulation is probably seriously affected by the presence of significant numbers of other 'contaminating' cell types. Several methodologies can be employed to enrich the target cells submitted for microarray analyses. These include magnetic sorting and laser capture microdissection. If a cell subpopulation of interest is small, very high-throughput cell separation technologies are needed to separate enough cells for conventional microarrays. 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. Since RNA rapidly degrades, and specific mRNAs turn over in minutes or hours, the cell separation process must be very rapid. Hence, high-throughput cell separation (HTS) 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 amplifie 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.......

【作者名称】: James F. Leary, Peter Szaniszlo, Tarl Prow, Lisa M. Reece, Nan Wang, David M. Asmuth
【作者单位】: University of Texas Medical Branch, Galveston, Texas 77555
【关 键 词】: high-throughput screening, flow cytometry/cell sorting, magnetic bead cell separation, gene expression microarrays
【会议名称】: Clinical Diagnostic Systems: Technologies and Instrumentation
【期刊论文数据库】: [DBS_Articles_01]
【期刊论文编号】: 100,988,734
【摘要长度】: 2,652
【会议地点】: San Jose, CA(US)
【会议组织】: University of Texas Medical Branch, Galveston, Texas 77555
【会议时间】: 2002
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