Transcript Profiling of Human Platelets Using Microarray and Serial Analysis of Gene Expression (SAGE)

  • Dmitri V. Gnatenko
  • John J. Dunn
  • John Schwedes
  • Wadie F. Bahou
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 496)


Platelets are anucleated cells that are generated from megakaryocytes via thrombopoiesis. They lack genomic DNA but have a pool of individual mRNA transcripts. Taken together, these mRNAs constitute a platelet transcriptome. Platelets have a unique and reproducible transcript profile, which includes ∼1,600–3,000 individual transcripts. In this chapter, we will focus on platelet purification and on transcript profiling using an Affymetrix microarray platform and serial analysis of gene expression (SAGE). Platelet purification is described in detail. Large-scale platelet purification schema is designed to purify platelets from apheresis platelet bags (∼3–5 × 1011 platelets/bag). Modification of this schema – small-scale platelet purification – is designed to isolate platelets from 20 ml of peripheral blood. This chapter provides detailed protocols for microarray and SAGE transcript profiling. We also discuss peculiarities of platelet purification, RNA isolation, and transcript profiling.

Key Words

Platelet RNA microarray serial analysis of gene expression 



This work was supported by NIH/NHLBI grants R21 HL076457 (D.V.G.) and HL086376 (W.F.B); Department of Defense grant MP048005 and a Targeted Research Award (D.V.G.) from Stony Brook University. Studies at the Brookhaven National Laboratory were supported by a Laboratory Directed Research and Development award (J.J.D.) and by the Offices of Biological and Environmental Research, and of Basic Energy Sciences (Division of Energy Biosciences) of the US Department of Energy.


  1. 1.
    Newman, P. J., Gorski, J., White, G. C., 2nd, et al. (1988) Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction. J Clin Invest 82, 739–743.CrossRefPubMedGoogle Scholar
  2. 2.
    Kieffer, N., Guichard, J., Farcet, J. P., et al. (1987) Biosynthesis of major platelet proteins in human blood platelets. Eur J Biochem 164, 189–195.CrossRefPubMedGoogle Scholar
  3. 3.
    Rinder, H., Schuster, J., Rinder, C., et al. (1998) Correlation of thrombosis with increased platelet turnover in thrombocytosis. Blood 91, 1288–1294.PubMedGoogle Scholar
  4. 4.
    Denis, M. M., Tolley, N. D., Bunting, M., et al. (2005) Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell 122, 379–391.CrossRefPubMedGoogle Scholar
  5. 5.
    Bugert, P., Dugrillon, A., Gunaydin, A., et al. (2003) Messenger RNA profiling of human platelets by microarray hybridization. Thromb Haemost 90, 738–748.PubMedGoogle Scholar
  6. 6.
    Gnatenko, D. V., Dunn, J. J., McCorkle, S. R., et al. (2003) Transcript profiling of human platelets using microarray and serial analysis of gene expression. Blood 101, 2285–2293.CrossRefPubMedGoogle Scholar
  7. 7.
    McRedmond, J. P., Park, S. D., Reilly, D. F., et al. (2004) Integration of proteomics and genomics in platelets: a profile of platelet proteins and platelet-specific genes. Mol Cell Proteomics 3, 133–144.PubMedGoogle Scholar
  8. 8.
    Gnatenko, D. V., Cupit, L. D., Huang, E. C., et al. (2005) Platelets express steroidogenic 17beta-hydroxysteroid dehydrogenases. Distinct profiles predict the essential thrombocythemic phenotype. Thromb Haemost 94, 412–421.PubMedGoogle Scholar
  9. 9.
    Hillmann, A. G., Harmon, S., Park, S. D., et al. (2006) Comparative RNA expression analyses from small-scale, single-donor platelet samples. J Thromb Haemost 4, 349–356.CrossRefPubMedGoogle Scholar
  10. 10.
    Shim, M. H., Hoover, A., Blake, N., et al. (2004) Gene expression profile of primary human CD34+CD38lo cells differentiating along the megakaryocyte lineage. Exp Hematol 32, 638–648.CrossRefPubMedGoogle Scholar
  11. 11.
    Tenedini, E., Fagioli, M. E., Vianelli, N., et al. (2004) Gene expression profiling of normal and malignant CD34-derived megakaryocytic cells. Blood 104, 3126–3135.CrossRefPubMedGoogle Scholar
  12. 12.
    Lockhart, D. J., Winzeler, E. A. (2000) Genomics, gene expression and DNA arrays. Nature 405, 827–836.CrossRefPubMedGoogle Scholar
  13. 13.
    Hoheisel, J. D. (2006) Microarray technology: beyond transcript profiling and genotype analysis. Nat Rev Genet 7, 200–210.CrossRefPubMedGoogle Scholar
  14. 14.
    Sausville, E. A., Holbeck, S. L. (2004) Transcription profiling of gene expression in drug discovery and development: the NCI experience. Eur J Cancer 40, 2544–2549.CrossRefPubMedGoogle Scholar
  15. 15.
    Gnatenko, D. V., Perrotta, P. L., Bahou, W. F. (2006) Proteomic approaches to dissect platelet function: one-half of the story. Blood 108, 3983–3991.CrossRefPubMedGoogle Scholar
  16. 16.
    Gnatenko, D. V., Bahou, W. F. (2006) Recent advances in platelet transcriptomics. Transfus Med Hemother 33, 217–226.CrossRefGoogle Scholar
  17. 17.
    Butte, A. (2002) The use and analysis of microarray data. Nat Rev Drug Discov 1,951–960.Google Scholar
  18. 18.
    Velculescu, V., Zhang, L., Vogelstein, B., et al. (1995) Serial analysis of gene expression. Science 270, 484–487.CrossRefPubMedGoogle Scholar
  19. 19.
    Velculescu, V., Zhang, L., Zhou, W. et al. (1997) Characterization of the yeast transcriptome. Cell 88, 243–251.CrossRefPubMedGoogle Scholar
  20. 20.
    Boyd, A. C., Charles, I. G., Keyte, J. W., et al. (1986) Isolation and computer-aided characterization of MmeI, a type II restriction endonuclease from Methylophilus methylotrophus. Nucleic Acids Res 14, 5255–5274.CrossRefPubMedGoogle Scholar
  21. 21.
    Tucholski, J., Skowron, P. M., Podhajska, A. J. (1995) MmeI, a class-IIS restriction endonuclease: purification and characterization. Gene 157, 87–92.CrossRefPubMedGoogle Scholar
  22. 22.
    Spinella, D. G., Bernardino, A. K., Redding, A. C., et al. (1999) Tandem arrayed ligation of expressed sequence tags (TALEST): a new method for generating global gene expression profiles. Nucleic Acids Res 27, e22.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Dmitri V. Gnatenko
    • 1
  • John J. Dunn
    • 2
  • John Schwedes
    • 3
  • Wadie F. Bahou
    • 1
  1. 1.Division of Hematology/Oncology Department of MedicineState University of New York at Stony BrookStony BrookUSA
  2. 2.Biology DepartmentBrookhaven National LaboratoryUptonUSA
  3. 3.University DNA Microarray Facility Office of Scientific Affairs, School of Medicine State University of New York at Stony BrookStony BrookUSA

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