BackgroundHuman blood platelets are essential to maintaining normal hemostasis, and platelet dysfunction often causes bleeding or thrombosis. Estimates of genome-wide platelet RNA expression using microarrays have provided insights to the platelet transcriptome but were limited by the number of known transcripts. The goal of this effort was to deep-sequence RNA from leukocyte-depleted platelets to capture the complex profile of all expressed transcripts.ResultsFrom each of four healthy individuals we generated long RNA (≥40 nucleotides) profiles from total and ribosomal-RNA depleted RNA preparations, as well as short RNA (<40 nucleotides) profiles. Analysis of ~1 billion reads revealed that coding and non-coding platelet transcripts span a very wide dynamic range (≥16 PCR cycles beyond β-actin), a result we validated through qRT-PCR on many dozens of platelet messenger RNAs. Surprisingly, ribosomal-RNA depletion significantly and adversely affected estimates of the relative abundance of transcripts. Of the known protein-coding loci, ~9,500 are present in human platelets. We observed a strong correlation between mRNAs identified by RNA-seq and microarray for well-expressed mRNAs, but RNASeq identified many more transcripts of lower abundance and permitted discovery of novel transcripts.ConclusionsOur analyses revealed diverse classes of non-coding RNAs, including: pervasive antisense transcripts to protein-coding loci; numerous, previously unreported and abundant microRNAs; retrotransposons; and thousands of novel un-annotated long and short intronic transcripts, an intriguing finding considering the anucleate nature of platelets. The data are available through a local mirror of the UCSC genome browser and can be accessed at: http://cm.jefferson.edu/platelets_2012/.

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Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stemloop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage-and/or tissue-specific miRNAs that are uncharacterized. SignificanceMicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology.

MicroRNAs (miRNAs) regulate cell physiology by altering protein expression, but the biology of platelet miRNAs is largely unexplored. We tested whether platelet miRNA levels were associated with platelet reactivity by genome-wide profiling using platelet RNA from 19 healthy subjects. We found that human platelets express 284 miRNAs. Unsupervised hierarchical clustering of miRNA profiles resulted in 2 groups of subjects that appeared to cluster by platelet aggregation phenotypes. Seventy-four miRNAs were differentially expressed (DE) between subjects grouped according to platelet aggregation to epinephrine, a subset of which predicted the platelet reactivity response. Using whole genome mRNA expression data on these same subjects, we computationally generated a highpriority list of miRNA-mRNA pairs in which the DE platelet miRNAs had binding sites in 3-untranslated regions of DE mRNAs, and the levels were negatively correlated. IntroductionOn rupture of atherosclerotic plaques, some persons form occlusive platelet thrombi whereas other persons repair the wound without occluding the vessel. The extreme interindividual variation in platelet reactivity probably contributes to the variation in both risk and clinical outcome of ischemic vascular disease because platelet hyper-reactivity has prospectively been shown to be a risk for recurrent coronary syndromes. 1 Although heritability strongly influences the interindividual variation in platelet reactivity, [2][3][4] there is a lack of understanding of the responsible genetic and molecular mechanisms. To understand better the basis for human platelet function, it is critical to define the genes that are expressed in the tissue of interest. We have previously used platelet RNA expression analyses from platelets of differing reactivity to identify differentially expressed (DE) platelet transcripts and proteins. 5 During the course of our studies, we found that a DE platelet microRNA (miRNA) altered the expression of VAMP8, a critical component of platelet granule exocytosis. miRNAs are small (ϳ 22 nucleotides) noncoding RNAs that function post-transcriptionally in regulating gene expression by inducing mRNA degradation or translation inhibition, generally by targeting the 3Ј-untranslated region (UTR) of mRNAs. 6 miRNAs were initially identified as regulators of genes involved in development but have since been shown to affect a broad range of normal physiologic processes, including hematopoietic lineage commitment, as well as pathologic conditions. 7,8 More than 1000 miRNAs have been identified, which are estimated to regulate most (Ͼ 60%) coding genes. 9 The cellular impact of most miRNA-mRNA interactions is a fine-tuning of protein output, and not a major repression of expression. 10 Importantly, as little as a 20% reduction in miRNA levels can produce a disease phenotype. 11 Recent data demonstrate a role for miRNAs in both normal and diseased human megakaryocytopoiesis. 8,12-17 Although we and others have observed miRNAs in platelets, 15,[18][19][20][21][22][23] th...

Aspirin-resistant patients as a group have reduced response to clopidogrel. Furthermore, we have identified a unique group of dual drug-resistant patients who may be at increased risk for thrombotic complications after PCI.