Comparative rna expression analyses from small-scale, single-donor platelet samples1

Abstract

Background: Comparisons of platelet RNAs could provide crucial information on platelet function, thrombopoiesis and the etiology of megakaryocyte (MK) or platelet disorders. Objectives: We developed a method for stringent purification of platelets from small blood samples from single donors. Purity of the platelet preparations was verified by an RT-PCR assay. We tested three methods to identify the differences in RNA between platelet sources. Methods: Differential hybridization to cDNA macro-arrays and suppressive-subtractive hybridization PCR (SSH-PCR) were used to compare RNAs from normal platelets to those from a Bernard-Soulier syndrome (BSS) patient. Affymetrix GeneChip U133 plus 2.0 arrays were used to compare male and female platelet RNAs. Results: Macroarrays identified similar to 7500 platelet transcripts, but failed to identify differentially expressed transcripts with confidence. SSH-PCR produced libraries almost exclusively of mitochondrial-derived transcripts, but included nuclear-encoded genes that could not be confirmed by immunoblotting of normal and BSS platelet lysates. The Affymetrix platform gave reproducible profiles from our small-scale purified platelet preparations, whereas a partially purified platelet preparation produced a drastically skewed transcript profile. The microarray analysis identified the heparanase precursor transcript as overexpressed in female platelets, and we observed variable yet consistently higher levels of heparanase protein in female platelets compared with male platelets in four independent donor pairs. Conclusions: This demonstrates for the first time that differential platelet transcript levels can identify changes in expression level of platelet proteins. Combined with our small-scale platelet preparation method, this establishes a system to compare platelets from the limited clinical sources to help elucidate molecular bases for platelet or megakaryocyte pathologies.