Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling by array of whole blood from sickle-cell disease patients using globin mRNA reduction

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Xiuli Xu

PROVIDER: E-GEOD-16728 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Characterization of whole blood gene expression profiles as a sequel to globin mRNA reduction in patients with sickle cell disease.

Raghavachari Nalini N Xu Xiuli X Munson Peter J PJ Gladwin Mark T MT

PloS one 20090803 8

Global transcriptome analysis of whole blood RNA using microarrays has been proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA. 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 accurately measure the steady state blood transcriptome in sickle cell patients we evaluated the efficacy of reduc ...[more]

PMID: 19649296

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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

Project description:Microarray-based gene expression analysis of peripheral whole blood is a common strategy in the development of clinically relevant biomarker panels for a variety of human diseases. However, the results of such an analysis are often plagued by decreased sensitivity and reliability due to the effects of relatively high levels of globin mRNA in whole blood. Globin reduction assays have been shown to overcome such effects, but they require large amounts of total RNA and may induce distinct gene expression profiles. The Illumina whole-genome DASL (WG-DASL) assay can detect gene expression levels using partially degraded RNA samples and has the potential to detect rare transcripts present in highly heterogeneous whole blood samples without the need for globin reduction. We therefore assessed the utility of the WG-DASL assay in the analysis of peripheral whole blood gene expression profiles. We find that gene expression detection is significantly increased with the use of WG-DASL compared to the standard in vitro transcription-based direct hybridization (IVT), while globin-probe-negative WG-DASL did not exhibit significant improvements over globin-probe-positive WG-DASL. Globin reduction increases the detection sensitivity and reliability of both WG-DASL and IVT with little effect on raw intensity correlations: raw intensity correlations between total RNA and globin-reduced RNA were 0.970 for IVT and 0.981 for WG-DASL. Overall, the detection sensitivity of the WG-DASL assay is higher than the IVT-based direct hybridization assay, with or without globin reduction, and should be considered in conjunction with globin reduction methods for future blood-based gene expression studies. Peripheral whole blood samples were collected from eight human donors in PAXGene tubes. RNA was isolated after freezing and storage, and then prepared for gene expression analysis using the Illumina Human-Ref8 v3.0 BeadChip. Alpha and beta globin were reduced from a portion of the total RNA using the GLOBINclear assay (Ambion, Austin, TX, USA). Two methods of microarray target preparation were examined: Illumina IVT-based direct hybridization (IVT) and Illumina Whole-Genome DASL (WG-DASL). Two DASL Assay Oligo pools (DAP) were utilized for DASL target preparation: the DASL Assay Oligo Pool with globin probes (DAP +) and the DASL Asssay Oligo Pool without globin probes (DAP-).

Project description:Background: Allergen inhalation challenge in mild asthmatics induces airflow obstruction, airway hyperresponsiveness and inflammation, providing a model for hypothesis-generating experiments to understand regulation of these responses. We have sought to evaluate the peripheral whole blood transcriptome, post-challenge compared to pre-challenge, and to determine the effect of globin mRNA reduction methodology. Methods: Asthmatic subjects (20-60 years of age, with stable, mild allergic asthma, n=17) underwent allergen inhalation challenges. All subjects had an early asthmatic response of ≥ 20% fall in FEV1; most had a late phase response of ≥ 15% fall in FEV1. Blood was collected immediately prior to, and two hours after allergen challenge. Transcriptome analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST arrays, with and without globin mRNA reduction (PAX-GR and PAX-NGR, respectively) in 4 subjects. Replication studies for expression of nine genes contributing to the top canonical pathway, Nrf2-mediated oxidative stress response pathway, were performed with 5 independent subjects with microarray data, also with the same 4 subjects had microarray results and 8 other independent subjects with QPCR. Data were analyzed using paired t-test and Partek® Genomics Suite™. Results: The number of differentially expressed probe sets from PAX-NGR samples was twice that of PAX-GR samples. Paired analysis of each subjects' pre- and post-sample additionally demonstrated twice the power to detect differentially expressed probe sets. The Nrf2-mediated oxidative stress response pathway was identified as the top canonical pathway in the PAX-NGR samples. ATP-binding cassette sub-family C member 1 (ABCC1) gene was significantly reduced two hours after allergen challenge in the 5 subjects’ microarray dataset and the 2 QPCR replication datasets with P < 0.05. Conclusions: Globin mRNA reduction does not provide benefits to detect differentially expressed genes during allergen inhalation challenge. Allergen inhalation challenge is associated with decreased peripheral blood cell transcript level of ABCC1 gene. 26 array were analyzed. We first identified differentially expreed genes between post- and pre-challenge samples from 4 asthmatic subjects by using globin reduction samples and non-globin reduction samples (16 arrays). Then we replicated our findings using 10 arrays from 5 asthmatic subjects challenged with allergen. Includes 4 normal blood samples.

Dataset Information

Transcription profiling by array of whole blood from sickle-cell disease patients using globin mRNA reduction

Publications

Characterization of whole blood gene expression profiles as a sequel to globin mRNA reduction in patients with sickle cell disease.

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