Comparative RNA expression analyses from small‐scale, single‐donor platelet samples

Abstract

Summary. 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 ∼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.

Human platelet microRNA-mRNA networks associated with age and gender revealed by integrated plateletomics

“…Our estimate of 7771 commonly expressed platelet genes was consistent with other reports of genome-wide microarray 27,31 and RNA-seq 16,18 analyses of smaller numbers of samples. The size of our dataset permits accurate estimates of platelet RNA levels in healthy subjects.…”

Sepsis Alters the Megakaryocyte–Platelet Transcriptional Axis Resulting in Granzyme B–mediated Lymphotoxicity

“…Thus, these previous studies assumed that changes in platelet function were at the posttranscriptional level. Platelets do contain reservoirs of mRNA, and a number of studies have reported the transcriptome of human platelets using mRNA profiling (11)(12)(13)(14). It has also been established that platelets regulate translation of their transcriptome in response to external stimuli (15)(16)(17).…”

Platelet RNA chips dip into thrombocytosis

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