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Integrative Flow Cytometric and Microarray Approaches for Use in Transcriptional Profiling

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Flow Cytometry Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 263))

Abstract

Flow cytometry and cell sorting provides unparalleled means for the identification and purification of specific cell types. It is a mature technology having been in existence commercially for the last 25 yr. High-throughput transcriptional profiling methods have emerged relatively recently. These provide the means to characterize efficiently the genome-wide contribution of individual genes to gene expression. A combination of these methods offers the opportunity to explore the relationship between gene expression and the ways in which different cell types are formed and maintained. This chapter provides a review of published methods for analysis of global gene expression within different cell types in complex tissues and organs, and provides practical details concerning microarray fabrication and use based on presynthesized 70-mer oligonucleotide array elements.

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References

  1. Loken, M. R. and Herzenberg, L. A. (1975) Analysis of cell-populations with a fluorescence-activated cell sorter. Ann. NY Acad. Sci. 254, 163–171.

    Article  PubMed  CAS  Google Scholar 

  2. De Rosa, S. C., Herzenberg, L. A., Herzenberg, L. A., and Roederer, M. (2001) Eleven-color, thirteen-parameter flow cytometry: Identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nat. Med. 7, 245–248.

    Article  PubMed  Google Scholar 

  3. Hara, M., Wang, X. Y., Kawamura, T, et al. (2003) Transgenic mice with green fluorescent protein-labeled pancreatic beta-cells. Am. J. Physiol. Endocrinol. Metab. 284, E177–E183.

    PubMed  CAS  Google Scholar 

  4. Galbraith, D. W., Bartos, J., and Dolezel, J. (2003) Flow cytometry and cell sorting in plant biotechnology, in Flow Cytometry in Biotechnology (Sklar, L., ed.), Oxford University Press, Oxford, UK.

    Google Scholar 

  5. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.

    Article  PubMed  CAS  Google Scholar 

  6. Tsien, R. (1998) The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544.

    Article  PubMed  CAS  Google Scholar 

  7. Lippincott-Schwartz, J. and Patterson, G. H. (2003) Development and use of fluorescent protein markers in living cells. Science 300, 87–91.

    Article  PubMed  CAS  Google Scholar 

  8. Haseloff, J., Siemering, K. R., Prasher, D. C., and Hodge, S. (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA 94, 2122–2127.

    Article  PubMed  CAS  Google Scholar 

  9. Chytilova, E., Macas, J., and Galbraith, D. W. (1999) Green fluorescent protein targeted to the nucleus, a transgenic phenotype useful for studies in plant biology. Ann. Botany 83, 645–654.

    Article  CAS  Google Scholar 

  10. Chytilova, E., Macas, J., Sliwinska, E., Rafelski, S., Lambert, G., and Galbraith, D. W. (2000) Nuclear dynamics in Arabidopsis thaliana. Mol. Biol. Cell 11, 2733–2741.

    PubMed  CAS  Google Scholar 

  11. Grebenok, R. J., Pierson, E. A., Lambert, G. M., et al. (1997) Green-fluorescent protein fusions for efficient characterization of nuclear localization signals. Plant Journal 11, 573–586.

    Article  PubMed  CAS  Google Scholar 

  12. Grebenok, R. J., Lambert, G. M., and Galbraith, D. W. (1997) Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants. Plant J. 12, 685–696.

    Article  CAS  Google Scholar 

  13. Galbraith, D. W. (2003) Global analysis of cell type-specific gene expression. Comp. Fund. Genomics 4, 208–215.

    Article  CAS  Google Scholar 

  14. Macas, J., Lambert, G. M., Dolezel, D., and Galbraith, D. W. (1998) NEST (Nuclear Expressed Sequence Tag) analysis: a novel means to study transcription through amplification of nuclear RNA. Cytometry 33, 460–468.

    Article  PubMed  CAS  Google Scholar 

  15. Hawley, T. S., Telford, W. G., and Hawley, R. G. (2001) “Rainbow” reporters for multispectral marking and lineage analysis of hematopoietic stem cells. Stem Cells 19, 118–124.

    Article  PubMed  CAS  Google Scholar 

  16. Hawley, T. S., Telford, W. G., Ramezani, A., and Hawley, R. G. (2001) Four-color flow cytometric detection of retrovirally expressed red, yellow, green and cyan fluorescent proteins. BioTechniques 30, 1028–1034.

    PubMed  CAS  Google Scholar 

  17. Meyer, K., Irminger, J.-C., Moss, L. G., et al. (1998) Sorting human β-cells consequent to targeted expression of green fluorescent protein. Diabetes 47, 1974–1977.

    Article  PubMed  CAS  Google Scholar 

  18. Keyoung, H. M., Roy, N. S., Benraiss, A., et al. (2001) High-yield selection and extraction of two promoter-defined phenotypes of neural stem cells from the fetal human brain. Nature Biotech. 19, 843–850.

    Article  CAS  Google Scholar 

  19. Roy, N. S., Wang, S., Harrison-Restelli, C., et al. (1999) Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter. J. Neurosci. 19, 9986–9995.

    PubMed  CAS  Google Scholar 

  20. Harkins, K. R., Jefferson, R. A., Kavanagh, T. A., Bevan, M. W., and Galbraith, D. W. (1990) Expression of photosynthesis-related gene fusions is restricted by cell-type in transgenic plants and in transfected protoplasts. Proc. Natl. Acad. Sci. USA 87, 816–820.

    Article  PubMed  CAS  Google Scholar 

  21. Sheen, J., Hwang, S., Niwa, Y., Kobayashi, H., and Galbraith, D. W. (1995) Green Fluorescent Protein as a new vital marker in plant cells. Plant J. 8, 777–784.

    Article  PubMed  CAS  Google Scholar 

  22. Nunes, M. C., Roy, N. S., Keyoung, H. M., et al. (2003) Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat. Med. 9, 439–447.

    Article  PubMed  CAS  Google Scholar 

  23. Roy, N. S., Wang, S., Jiang, L., et al. (2000) Promoter-selected selection and isolation of neural progenitor cells from the adult human ventricular zone. J. Neurosci. Res. 59, 321–331.

    Article  PubMed  CAS  Google Scholar 

  24. Sawamoto, K., Nakao, N., Kobayashi, K., et al. (2001) Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons. Proc. Natl. Acad. Sci. USA 98, 6423–6428.

    Article  PubMed  CAS  Google Scholar 

  25. Malatesta, P., Hartfuss, E., and Gotz, M. (2000) Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage. Development 127, 5253–5263.

    PubMed  CAS  Google Scholar 

  26. Takeuchi, Y., Yoshizaki, G., Kobayashi, T., and Takeuchi, T. (2002) Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter. Biol. Reprod. 67, 1087–1092.

    Article  PubMed  CAS  Google Scholar 

  27. Fei, Y. J. and Hughes, T. E. (2001) Transgenic expression of the jellyfish green fluorescent protein in the cone photoreceptors of the mouse. Visual Neurosci. 18, 615–623.

    Article  CAS  Google Scholar 

  28. Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470.

    Article  PubMed  CAS  Google Scholar 

  29. Xu, W., Bak, S., Decker, A., Paquette, S. M., Feyereisen, R., and Galbraith D. W. (2001) Microarray-based analysis of gene expression in very large gene families: the Cytochrome P450 gene superfamily of Arabidopsis thaliana. Gene 272, 61–74.

    Article  PubMed  CAS  Google Scholar 

  30. Galbraith, D. W. and Lucretti, S. (2000) Large particle sorting in Flow Cytometry and Cell Sorting, 2nd edit. (Radbruch, A., ed.), Springer-Verlag, Berlin, pp. 293–317.

    Google Scholar 

  31. Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., and Eberwine, J. H. (1990) Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA 87, 1663–1667.

    Article  PubMed  Google Scholar 

  32. Gubler, U. and Hoffman, B. J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263–269.

    Article  PubMed  CAS  Google Scholar 

  33. Wang, E., Miller, L. D., Ohnmacht, G. A., Liu, E. T., and Marincola, F. M. (2000) High-fidelity mRNA amplification for gene profiling. Nat. Biotechnol. 18, 457–459.

    Article  PubMed  CAS  Google Scholar 

  34. Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001) Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res. 29, E29.

    Article  PubMed  CAS  Google Scholar 

  35. Zhao, H., Hastie, T., Whitfield, M. L., Borresen-Dale, A. L., and Jeffrey, S. S. (2002) Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis. B.M.C. Genomics 3, 31.

    Article  CAS  Google Scholar 

  36. Luzzi, V., Mahadevappa, M., Raja, R., Warrington, J. A., and Watson, M. A. (2003) Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis. J. Mol. Diagn. 5, 9–14.

    Article  PubMed  CAS  Google Scholar 

  37. Xiang, C. C., Chen, M., Ma, L., et al. (2003) A new strategy to amplify degraded RNA from small tissue samples for microarray studies. Nucleic Acids Res. 31, e53.

    Article  PubMed  Google Scholar 

  38. Iscove, N. N., Barbara, M., Gu, M., Gibson, M., Modi, C., and Winegarden, N. (2002) Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA. Nat. Biotechnol. 20, 940–943.

    Article  CAS  Google Scholar 

  39. Brandt, S., Kloska, S., Altmann, T., and Kehr, J. (2002) Using array hybridization to monitor gene expression at the single cell level. J. Exp. Bot. 53, 2315–2323.

    Article  PubMed  CAS  Google Scholar 

  40. Ginsberg, S. D. and Che, S. (2002) RNA amplification in brain tissues. Neurochem. Res. 27, 981–992.

    Article  PubMed  CAS  Google Scholar 

  41. Aoyagi, K., Tatsuta, T., Nishigaki, M., et al. (2003) A faithful method for PCR-mediated global mRNA amplification and its integration into microarray analysis on laser-captured cells. Biochem. Biophys. Res. Commun. 300, 915–920.

    Article  PubMed  CAS  Google Scholar 

  42. Chiang, M. K. and Melton, D. A. (2003) Single-cell transcript analysis of pancreas development. Dev. Cell 4, 383–393.

    Article  PubMed  CAS  Google Scholar 

  43. Seth, D., Gorrell, M. D., McGuinness, P. H., et al. (2003) SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates. J. Biochem. Biophys. Methods 55, 53–66.

    Article  PubMed  CAS  Google Scholar 

  44. Smith, L., Underhill, P., Pritchard, C., et al. (2003) Single primer amplification (SPA) of cDNA for microarray expression analysis. Nucleic Acids Res. 31, e9.

    Article  PubMed  Google Scholar 

  45. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal. Biochem. 162, 156–159.

    Article  PubMed  CAS  Google Scholar 

  46. Sambrook, J. and Russel, D. W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  47. Birnbaum, K., Shasha, D. E., Wang, J. Y., et al. (2003) A gene expression map of the Arabidopsis root. Science 302, 1956–1960.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Plant Sciences, University of Arizona, Tucson, AZ

    David W. Galbraith, Rangasamy Elumalai & Fang Cheng Gong

Authors
  1. David W. Galbraith
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  2. Rangasamy Elumalai
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  3. Fang Cheng Gong
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Editor information

Editors and Affiliations

  1. American Red Cross, Rockville, MD

    Teresa S. Hawley  & Robert G. Hawley  & 

  2. The George Washington University, Washington, DC

    Robert G. Hawley

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© 2004 Humana Press Inc.

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Galbraith, D.W., Elumalai, R., Gong, F.C. (2004). Integrative Flow Cytometric and Microarray Approaches for Use in Transcriptional Profiling. In: Hawley, T.S., Hawley, R.G. (eds) Flow Cytometry Protocols. Methods in Molecular Biology™, vol 263. Humana Press. https://doi.org/10.1385/1-59259-773-4:259

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  • DOI: https://doi.org/10.1385/1-59259-773-4:259

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-234-6

  • Online ISBN: 978-1-59259-773-4

  • eBook Packages: Springer Protocols

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