Genome-wide microRNA analysis of reticulocytes isolated from patients with sickle cell disease
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PROVIDER: PRJNA436707 | ENA |
REPOSITORIES: ENA
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Project description:MiRNAs are non-protein-coding small RNA molecules negatively regulating gene through inhibition of mRNA. The characteristics of microRNA expression patterns obtained from sickle cell diseases are not clearly elucidated. In this study, we recruited 12 children and adults suffering from sickle cells diseases with high HbF fetal hemoglobin (HbF) group (6 subjects) and lower HbF (6 subjects). The peripheral blood mononuclear cell (PBMN) were processed using a MACS column with magnetic anti-CD71 antibody to isolate reticulocytes according to the manufacturer’s instructions (Miltenyi Biotec, San Diego, CA). After data normalization, 327 miRNA were identified by PCA (principal component analysis) as differentially expressed in low HbF group compared to high HbF groups.
2018-03-03 | GSE111356 | GEO
Project description:Background Since mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS). Methods and Findings Using a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation. Conclusions In summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases. Keywords: Sickle cell disease RBC profiling The microRNA of purified erythrocytes from 7 normal and 12 HbSS individuals
2009-04-04 | E-GEOD-11060 | biostudies-arrayexpress
Project description:Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation . In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations. Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable. Keywords: Microarrays, PAXgene, globin reduction, whole blood, PBMC There are 10 samples for each of PBMC, PAX and PAX globin-reduced, where 5 samples come from sickle-cell patients and 5 from healthy controls.
2009-06-20 | E-GEOD-16728 | biostudies-arrayexpress
Project description:Background Since mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS). Methods and Findings Using a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation. Conclusions In summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases. Keywords: Sickle cell disease RBC profiling
2009-04-04 | GSE11060 | GEO
Project description:Circulating platelets from Sickle cell disease (SCD) patients express distinct gene expression patterns that regulate function. The objective of this study is to identify a role of post-transcriptional regulation of the platelet transcriptional signaling by microRNAs. Comparison of microRNA expression in platelets from SCD patients and control subjects, from 2 cohorts-University of Pittsburgh and National Institutes of Health.
2013-06-20 | E-GEOD-41574 | biostudies-arrayexpress
Project description:Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation . In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations. Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable. Keywords: Microarrays, PAXgene, globin reduction, whole blood, PBMC
2009-06-20 | GSE16728 | GEO
Project description:To analyze expression of inflammatory cytokines in Exhaled Breath Condensates from pediatric patients with sickle cell disease, asthma, sickle cell disease and asthma, and controls
2020-05-08 | GSE150056 | GEO
Project description:Circulating platelets from Sickle cell disease (SCD) patients express distinct gene expression patterns that regulate function. The objective of this study is to identify a role of post-transcriptional regulation of the platelet transcriptional signaling by microRNAs.
2013-06-20 | GSE41574 | GEO
Project description:Sickle cell disease (SCD) is a devastating hemoglobinopathy prevalent in Chhattisgarh and other states of central India. Clinical features in SCD arise mainly due to anemia and vaso-occlusion and inflammation leading to gradual multiple organ failure. Vaso-occlusive crisis (VOC) is a common cause of sudden death among SCD patients. Aim of the study was to evaluate gene expression in patients of SCD in a quest to search up-regulated genes in VOC.
2017-06-30 | GSE72999 | GEO
Project description:In sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide (NO) and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, suggesting that pathological platelet activation may contribute to sickle cell disease vasculopathy. Studies were therefore undertaken to examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate here the feasibility of comparative platelet transcriptome studies on clinical samples from single donors, by the application of RNA amplification followed by microarray-based analysis of 54,000 probe sets. Data mining an existing microarray database, we identified 220 highly abundant genes in platelets and a subset of 72 relatively platelet-specific genes, defined by more than 10-fold increased expression compared to the median of other cell types in the database with amplified transcripts. The highly abundant platelet transcripts found in the current study included 82% or 70% of platelet abundant genes identified in two previous gene expression studies on non-amplified mRNA from pooled or apheresis samples, respectively. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to 12 African American controls, at a 3-fold cut-off and 5% false discovery rate, we identified ~100 differentially expressed genes, including multiple genes involved in arginine metabolism and redox homeostasis. Further characterization of these pathways using real time PCR and biochemical assays revealed increased arginase II expression and activity and decreased platelet polyamine levels. These studies suggest a potential pathogenic role for platelet arginase and altered arginine and polyamine metabolism in sickle cell disease and provide a novel framework for the study of disease-specific platelet biology. Keywords: Platelets, genes, enzymes, metabolism, signal transduction, sickle cell disease
2008-05-22 | GSE11524 | GEO