Received on April 7, 2004
Accepted on September 13, 2004

Hemostasis and Thrombosis

Gene Expression Analysis in Platelets from a Single Donor: Evaluation of a PCR-Based Amplification Technique

¹ Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Germany, and These authors contributed equally to this work
² Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessen, University of Heidelberg, Faculty of Clinical Medicine, Mannheim, Germany, and These authors contributed equally to this work
³ Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Germany
⁴ Department of Haemostaseology, Clinical Immunology and Transfusion Medicine, Kerckhoff-Klinik, Bad Nauheim, Germany
⁵ Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessen, University of Heidelberg, Faculty of Clinical Medicine, Mannheim, Germany

Background: Genetic analysis of platelet mRNA may facilitate the diagnosis of disorders affecting the megakaryocytic-platelet lineage. Its use, however, is limited by the exceptionally small yield of platelet mRNA and the risk of leukocyte contamination during platelet preparation.

Methods: We depleted platelet suspensions of leukocytes by filtration and used a PCR-based RNA amplification step [switching mechanism at the 5' end of RNA templates (SMART)]. We tested the reliability and precision of the RNA amplification procedure by use of real-time PCR to measure quantities of specific transcripts: von Willebrand factor (vWF), A-subunit of coagulation factor XIII (F13A), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Microarray analysis was performed on platelet RNA with and without amplification.

Results: Microgram quantities of platelet-specific cDNAs were produced from as little as 50 ng of total platelet RNA or 40 mL of whole blood. At cycle numbers <16, amplification of all transcripts tested was exponential with slightly more efficient amplification of low-abundance transcripts. Expression profiling of 9850 genes gave identical results for 9815 genes (1576 positive/8239 negative). Eight transcripts failed to be amplified by the SMART procedure. Expression of vWF, F13A, and GAPDH transcripts showed only minor day-to-day variations in three healthy individuals.

Conclusion: The proposed protocol makes extremely small amounts of platelet RNA available for gene expression analysis in single patients.