The importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics

James F. Leary, Peter Szaniszlo, Tarl Prow, Lisa M. Reece, Nan Wang, David M. Asmuth

Research output: Chapter in Book/Report/Conference proceedingConference contribution

4 Citations (Scopus)

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. 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 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.

Original languageEnglish (US)
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
EditorsG.E. Cohn
Pages1-8
Number of pages8
Volume4625
DOIs
StatePublished - 2002
EventClinical Diagnostic Systems: Technologies and Instrumentation - San Jose, CA, United States
Duration: Jan 22 2002Jan 24 2002

Other

OtherClinical Diagnostic Systems: Technologies and Instrumentation
CountryUnited States
CitySan Jose, CA
Period1/22/021/24/02

Fingerprint

Gene expression
Microarrays
Throughput
cells
gene expression
Sorting
Amplification
Microdissection
Genes
Cells
profiles
Flow cytometry
Genomics
Proteomics
Transcription
RNA
Purification
classifying
Messenger RNA
genes

Keywords

  • Flow cytometry/cell sorting
  • Gene expression microarrays
  • High-throughput screening
  • Magnetic bead cell separation

ASJC Scopus subject areas

  • Electrical and Electronic Engineering
  • Condensed Matter Physics

Cite this

Leary, J. F., Szaniszlo, P., Prow, T., Reece, L. M., Wang, N., & Asmuth, D. M. (2002). The importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics. In G. E. Cohn (Ed.), Proceedings of SPIE - The International Society for Optical Engineering (Vol. 4625, pp. 1-8) https://doi.org/10.1117/12.469773

The importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics. / Leary, James F.; Szaniszlo, Peter; Prow, Tarl; Reece, Lisa M.; Wang, Nan; Asmuth, David M.

Proceedings of SPIE - The International Society for Optical Engineering. ed. / G.E. Cohn. Vol. 4625 2002. p. 1-8.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Leary, JF, Szaniszlo, P, Prow, T, Reece, LM, Wang, N & Asmuth, DM 2002, The importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics. in GE Cohn (ed.), Proceedings of SPIE - The International Society for Optical Engineering. vol. 4625, pp. 1-8, Clinical Diagnostic Systems: Technologies and Instrumentation, San Jose, CA, United States, 1/22/02. https://doi.org/10.1117/12.469773
Leary JF, Szaniszlo P, Prow T, Reece LM, Wang N, Asmuth DM. The importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics. In Cohn GE, editor, Proceedings of SPIE - The International Society for Optical Engineering. Vol. 4625. 2002. p. 1-8 https://doi.org/10.1117/12.469773
Leary, James F. ; Szaniszlo, Peter ; Prow, Tarl ; Reece, Lisa M. ; Wang, Nan ; Asmuth, David M. / The importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics. Proceedings of SPIE - The International Society for Optical Engineering. editor / G.E. Cohn. Vol. 4625 2002. pp. 1-8
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