In:
Clinical Chemistry, Oxford University Press (OUP), Vol. 50, No. 12 ( 2004-12-01), p. 2271-2278
Kurzfassung:
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 & lt;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.
Materialart:
Online-Ressource
ISSN:
0009-9147
,
1530-8561
DOI:
10.1373/clinchem.2004.035386
Sprache:
Englisch
Verlag:
Oxford University Press (OUP)
Publikationsdatum:
2004
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