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Transcript profiling of human platelets using microarray and serial analysis of gene expression (SAGE)

Dmitri V Gnatenko1, John J Dunn, John Schwedes

  • 1Division of Hematology/Oncology, Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY, USA.

Methods in Molecular Biology (Clifton, N.J.)
|October 8, 2008
PubMed

Insights

Platelets, lacking DNA, possess a unique transcriptome of 1,600-3,000 mRNAs. This study details methods for platelet purification and transcript profiling using microarray and SAGE techniques.

Area of Science:

  • Hematology
  • Molecular Biology
  • Genomics

Background:

  • Platelets are anucleated cells derived from megakaryocytes through thrombopoiesis.
  • Despite lacking genomic DNA, platelets contain a substantial pool of mRNA transcripts, forming a platelet transcriptome.
  • This platelet transcriptome exhibits a unique and reproducible profile, comprising approximately 1,600-3,000 distinct mRNA transcripts.

Purpose of the Study:

  • To provide detailed protocols for platelet purification.
  • To describe methods for transcript profiling in platelets using specific platforms.
  • To discuss the unique considerations in platelet purification, RNA isolation, and transcript analysis.

Main Methods:

  • Detailed protocols for large-scale platelet purification from apheresis bags (3-5 x 10^11 platelets/bag).
  • Modified small-scale platelet purification protocols for isolating platelets from 20 ml of peripheral blood.
  • Transcript profiling using Affymetrix microarray and Serial Analysis of Gene Expression (SAGE) platforms.

Main Results:

  • Established reproducible protocols for purifying platelets from various blood volumes.
  • Successfully applied microarray and SAGE for comprehensive platelet transcriptome analysis.
  • Identified and characterized the unique mRNA profile within platelets.

Conclusions:

  • Platelet purification and transcript profiling are feasible using the described methodologies.
  • The characterized platelet transcriptome provides a basis for understanding platelet function and biology.
  • Detailed protocols facilitate further research into platelet mRNA content and its implications.

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