Gene expression analysis in platelets from a single donor: evaluation of a PCR-based amplification technique.
Clin Chem
; 50(12): 2271-8, 2004 Dec.
Article
en En
| MEDLINE
| ID: mdl-15472037
ABSTRACT
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.
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Bases de datos:
MEDLINE
Asunto principal:
Plaquetas
/
Perfilación de la Expresión Génica
Tipo de estudio:
Guideline
Límite:
Humans
Idioma:
En
Revista:
Clin Chem
Asunto de la revista:
QUIMICA CLINICA
Año:
2004
Tipo del documento:
Article
País de afiliación:
Alemania