Project description:Sickle cell disease is associated with systemic complications, many associated with either severity of disease or increased risk of mortality. We sought to identify a circulating gene expression profile whose predictive capacity spanned the spectrum of these poor outcomes in sickle cell disease. The Training cohort consisted of patients with SCD who were prospectively recruited from the University of Illinois. The Testing cohort consisted of a combination of patients prospectively seen at two separate institutions including the University of Chicago and Howard University.
Project description:Sickle cell disease is associated with systemic complications, many associated with either severity of disease or increased risk of mortality. We sought to identify a circulating gene expression profile whose predictive capacity spanned the spectrum of these poor outcomes in sickle cell disease. The Training cohort consisted of patients with SCD who were prospectively recruited from the University of Illinois. The Testing cohort consisted of a combination of patients prospectively seen at two separate institutions including the University of Chicago and Howard University.
Project description:Sickle cell disease is associated with systemic complications, many associated with either severity of disease or increased risk of mortality. We sought to identify a circulating gene expression profile whose predictive capacity spanned the spectrum of these poor outcomes in sickle cell disease. The Training cohort consisted of patients with SCD who were prospectively recruited from the University of Illinois. The Testing cohort consisted of a combination of patients prospectively seen at two separate institutions including the University of Chicago and Howard University
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.
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.