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

Raghavachari N, Xu X, Munson PJ, Gladwin MT - PLoS ONE (2009)

Bottom Line: In order to accurately measure the steady state blood transcriptome in sickle cell patients we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA for genome-wide transcriptome analyses using microarrays.This led to an improvement in microarray data quality by reducing data variability, with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations.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.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary and Vascular Medicine Branch, National Heart Lung and Blood Institute, NIH, Bethesda, MD, USA. nraghavachari@nhlbi.nih.gov

ABSTRACT
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 reducing globin transcripts in PAXgene stabilized RNA for genome-wide transcriptome analyses using microarrays. 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 by reducing data variability, with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. Analysis of differences between the whole blood transcriptome and PBMC transcriptome revealed 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.

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A. Expression of 18s, globin transcripts α, β by RT-PCR in PAX and PAX-GR from control and SCD samples. Ct values in the expression of globins are given as mean±SD for each group. Red bar - PAX-Control; Green bar - PAX-GR Control; Yellow bar - PAX-SCD; Blue bar - PAX-GR-SCD. B. Normalized signal intensities for β globin probe sets from microarrays for control and SCD samples. Values are given as mean±SD. Grey bar - PAX; Black bar - PAX-GR. C. Normalized signal intensities for α globin probesets from microarrays for control and SCD samples. Values are given as mean±SD. Grey bar - PAX; Black bar - PAX-GR.
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pone-0006484-g002: A. Expression of 18s, globin transcripts α, β by RT-PCR in PAX and PAX-GR from control and SCD samples. Ct values in the expression of globins are given as mean±SD for each group. Red bar - PAX-Control; Green bar - PAX-GR Control; Yellow bar - PAX-SCD; Blue bar - PAX-GR-SCD. B. Normalized signal intensities for β globin probe sets from microarrays for control and SCD samples. Values are given as mean±SD. Grey bar - PAX; Black bar - PAX-GR. C. Normalized signal intensities for α globin probesets from microarrays for control and SCD samples. Values are given as mean±SD. Grey bar - PAX; Black bar - PAX-GR.

Mentions: To confirm the effectiveness of globin mRNA reduction, RNA samples from both PAX and PAX-GR samples were analyzed by RT-PCR using taqman primers for both alpha and beta globins and the normalized CT values were calculated using 18s as the house keeping gene. There was significant depletion of both the alpha and beta globins in PAX-GR samples as represented by the increase in delta CT values (normalized to 18S RNA) for both beta and alpha as a sequel to globin reduction as depicted in Figure 2A. Gamma globins did not show a significant difference between the PAX and PAX-GR samples. Examination of the S10 transformed signal intensity for the alpha and beta globins in the PAX and PAX-GR samples showed a similar pattern in the globin expression level with a signal intensity difference of >0.3 (or approximately log10 2 fold representing about 3 fold on the S10 transform scale) between the PAX and PAX-GR for both the alpha and beta globin genes. (Figures 2B & C).


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

Raghavachari N, Xu X, Munson PJ, Gladwin MT - PLoS ONE (2009)

A. Expression of 18s, globin transcripts α, β by RT-PCR in PAX and PAX-GR from control and SCD samples. Ct values in the expression of globins are given as mean±SD for each group. Red bar - PAX-Control; Green bar - PAX-GR Control; Yellow bar - PAX-SCD; Blue bar - PAX-GR-SCD. B. Normalized signal intensities for β globin probe sets from microarrays for control and SCD samples. Values are given as mean±SD. Grey bar - PAX; Black bar - PAX-GR. C. Normalized signal intensities for α globin probesets from microarrays for control and SCD samples. Values are given as mean±SD. Grey bar - PAX; Black bar - PAX-GR.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2714456&req=5

pone-0006484-g002: A. Expression of 18s, globin transcripts α, β by RT-PCR in PAX and PAX-GR from control and SCD samples. Ct values in the expression of globins are given as mean±SD for each group. Red bar - PAX-Control; Green bar - PAX-GR Control; Yellow bar - PAX-SCD; Blue bar - PAX-GR-SCD. B. Normalized signal intensities for β globin probe sets from microarrays for control and SCD samples. Values are given as mean±SD. Grey bar - PAX; Black bar - PAX-GR. C. Normalized signal intensities for α globin probesets from microarrays for control and SCD samples. Values are given as mean±SD. Grey bar - PAX; Black bar - PAX-GR.
Mentions: To confirm the effectiveness of globin mRNA reduction, RNA samples from both PAX and PAX-GR samples were analyzed by RT-PCR using taqman primers for both alpha and beta globins and the normalized CT values were calculated using 18s as the house keeping gene. There was significant depletion of both the alpha and beta globins in PAX-GR samples as represented by the increase in delta CT values (normalized to 18S RNA) for both beta and alpha as a sequel to globin reduction as depicted in Figure 2A. Gamma globins did not show a significant difference between the PAX and PAX-GR samples. Examination of the S10 transformed signal intensity for the alpha and beta globins in the PAX and PAX-GR samples showed a similar pattern in the globin expression level with a signal intensity difference of >0.3 (or approximately log10 2 fold representing about 3 fold on the S10 transform scale) between the PAX and PAX-GR for both the alpha and beta globin genes. (Figures 2B & C).

Bottom Line: In order to accurately measure the steady state blood transcriptome in sickle cell patients we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA for genome-wide transcriptome analyses using microarrays.This led to an improvement in microarray data quality by reducing data variability, with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations.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.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary and Vascular Medicine Branch, National Heart Lung and Blood Institute, NIH, Bethesda, MD, USA. nraghavachari@nhlbi.nih.gov

ABSTRACT
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 reducing globin transcripts in PAXgene stabilized RNA for genome-wide transcriptome analyses using microarrays. 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 by reducing data variability, with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. Analysis of differences between the whole blood transcriptome and PBMC transcriptome revealed 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.

Show MeSH
Related in: MedlinePlus