Skip to main content

Genome-Wide Platelet RNA Profiling in Clinical Samples

  • Protocol
DNA and RNA Profiling in Human Blood

Part of the book series: METHODS IN MOLECULAR BIOLOGY™ ((MIMB,volume 496))

Abstract

Human blood platelets are anucleate cells that contain minute amounts of translational active mRNA. Investigation of the gene expression profile by microarray analysis has become an excellent tool for better understanding of normal and pathological platelet function. Its use, however, is often limited by the low yield of megakaryocytic-derived mRNA, the possible contamination with leukocytes during platelet preparation and the small platelet volume in clinical settings, especially in pediatric patients. In this chapter, we present a protocol for the isolation of leukocyte-depleted platelet samples in clinical settings and an optimized procedure for transcript profiling, using the Agilent oligo microarray technology. In particular, we discuss the special aspects of platelet purification when working with blood sample volumes not exceeding 3–5 ml, which is typical in pediatric patients and we furthermore provide detailed information for transcript profiling of extremely small amounts of platelet RNA.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ault, K. A., Knowles, C. (1995) In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation. Exp Hematol 23, 996–1001.

    CAS  PubMed  Google Scholar 

  2. Kienast. J., Schmitz, G., (1990) Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Blood 75, 116–121.

    CAS  PubMed  Google Scholar 

  3. 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.

    Article  CAS  PubMed  Google Scholar 

  4. Bugert, P., Dugrillon, A., Gunaydin, A., et al. (2003) Messenger RNA profiling of human platelets by microarray hybridization. Thromb Haemost 90, 738–748.

    CAS  PubMed  Google Scholar 

  5. Fink, L., Holschermann, H., Kwapiszewska, G., et al. (2003) Characterization of platelet-specific mRNA by real-time PCR after laser-assisted microdissection. Thromb Haemost 90, 749–756.

    CAS  PubMed  Google Scholar 

  6. Rolf, N., Knoefler, R., Suttorp, M., et al. (2005) Optimized procedure for platelet RNA profiling from blood samples with limited platelet numbers. Clin Chem 51, 1078–1080.

    Article  CAS  PubMed  Google Scholar 

  7. Matz, M., Shagin, D., Bogdanova, E., et al. (1999) Amplifikation of cDNA ends based on template switching effect and step-out PCR. Nucleic Acids Res 27, 1558–60.

    Article  CAS  PubMed  Google Scholar 

  8. Schwabe, H., Stein, U., Walther, W. (2000) High-copy cDNA amplification of minimal total RNA quantities for gene expression analysis. Mol Biotechnol 14. 165–172.

    Article  CAS  PubMed  Google Scholar 

  9. Zhu, Y. Y., Machleder, E. M., Chenchik, A., et al. (2001) Reverse transcription template switching: a SMART approach for full length cDNA library construction. Biotechniques 30(4), 892–897.

    CAS  PubMed  Google Scholar 

  10. Phillips, J., Eberwine, J. H. (1996) Antisense RNA amplification: A linear amplification method for analyzing the mRNA population from single living cells. Methods 10(3),283–288.

    Article  CAS  PubMed  Google Scholar 

  11. Baugh, L. R., Hill, A. A., Brown, E. L., et al. (2001) Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29(5), E29.

    Article  CAS  PubMed  Google Scholar 

  12. Hu, L., Wang, J., Baggerly, K., et al. (2002) Obtaining reliable information from minute amounts of RNA using cDNA microarrays. BMC Genomics 3, 16.

    Article  PubMed  Google Scholar 

  13. Rox, M. J., Bugert, P., Müller, J., et al. (2004) Gene expression analysis in platelets from a single donor: Evaluation of a PCR-based amplification technique. Clin Chem 50(12), 2271–2278.

    Article  CAS  PubMed  Google Scholar 

  14. Schlenke, P. (2005) Leukocyte reduction in blood component supply: the impact of flow cytometry in assessing residual leukocytes. Transfus Med Hemother 32, 12–19.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Cite this protocol

Schedel, A., Rolf, N. (2009). Genome-Wide Platelet RNA Profiling in Clinical Samples. In: Bugert, P. (eds) DNA and RNA Profiling in Human Blood. METHODS IN MOLECULAR BIOLOGY™, vol 496. Humana Press. https://doi.org/10.1007/978-1-59745-553-4_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-553-4_17

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-93-0

  • Online ISBN: 978-1-59745-553-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics