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| Status |
Public on Jun 20, 2009 |
| Title |
Characterization of whole blood gene expression profiles in sickle-cell disease patients using globin mRNA reduction |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
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
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| Overall design |
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.
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| Contributor(s) |
Raghavachari N, Xu X, Cooper RA, Munson PJ, Gladwin MT |
| Citation(s) |
19649296 |
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| Submission date |
Jun 19, 2009 |
| Last update date |
Mar 25, 2019 |
| Contact name |
Xiuli Xu |
| E-mail(s) |
[email protected]
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| Phone |
301-402-4263
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| Organization name |
NHLBI, NIH
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| Department |
VMB,
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| Lab |
Microarray Core
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| Street address |
Building 10-CRC, Room 5-5140
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892-1454 |
| Country |
USA |
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| Platforms (1) |
| GPL570 |
[HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array |
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| Samples (30)
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| Relations |
| BioProject |
PRJNA117495 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE16728_RAW.tar |
131.6 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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