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

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.

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.

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.

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