Gene expression analysis in platelets from a single donor: evaluation of a PCR-based amplification technique.
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.


CONCLUSIONS
The proposed protocol makes extremely small amounts of platelet RNA available for gene expression analysis in single patients.
Rox
Jutta Maria
JM
Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Bonn, Germany.
Bugert
Peter
P
Müller
Jens
J
Schorr
Alexander
A
Hanfland
Peter
P
Madlener
Katharina
K
Klüter
Harald
H
Pötzsch
Bernd
B
D001792:Blood Platelets / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D047589:Leukocyte Reduction Procedures; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012313:RNA