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Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA.

Mastrokolias A, den Dunnen JT, van Ommen GB, 't Hoen PA, van Roon-Mom WM - BMC Genomics (2012)

Bottom Line: Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples.In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples.The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Human and Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, 2333ZC, Leiden, The Netherlands.

ABSTRACT

Background: Transcriptome analysis is of great interest in clinical research, where significant differences between individuals can be translated into biomarkers of disease. Although next generation sequencing provides robust, comparable and highly informative expression profiling data, with several million of tags per blood sample, reticulocyte globin transcripts can constitute up to 76% of total mRNA compromising the detection of low abundant transcripts. We have removed globin transcripts from 6 human whole blood RNA samples with a human globin reduction kit and compared them with the same non-reduced samples using deep Serial Analysis of Gene Expression.

Results: Globin tags comprised 52-76% of total tags in our samples. Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples. In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples. Removing globin transcripts allowed us to also identify 2112 genes that could not be detected in the non-globin reduced samples, with roles in cell surface receptor signal transduction, G-protein coupled receptor protein signalling pathways and neurological processes.

Conclusions: The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

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Related in: MedlinePlus

Differential expression of non-globin reduced versus globin reduced whole blood samples. A. MA-plot (log concentration on x-axis, log fold-change on y-axis) showing that the majority of differentially expressed genes (P value < 0.01, indicated in red) are up-regulated while the 4 globin genes showed the strongest reduction. Genes with zero expression in one of the groups are shown on the left end of the plot. HBD: hemoglobin delta, HBB: hemoglobin beta, HBA1: hemoglobin alpha 1, HBA1: hemoglobin alpha 2. CXorf25 has a logConc of -18.79 and a logFC of -14.3 and is not shown on this graph. B. Heatmap of normalised expression values of 21633 genes across the 6 non reduced and 6 reduced (R) samples where green depicts high expression and red low expression values.
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Figure 2: Differential expression of non-globin reduced versus globin reduced whole blood samples. A. MA-plot (log concentration on x-axis, log fold-change on y-axis) showing that the majority of differentially expressed genes (P value < 0.01, indicated in red) are up-regulated while the 4 globin genes showed the strongest reduction. Genes with zero expression in one of the groups are shown on the left end of the plot. HBD: hemoglobin delta, HBB: hemoglobin beta, HBA1: hemoglobin alpha 1, HBA1: hemoglobin alpha 2. CXorf25 has a logConc of -18.79 and a logFC of -14.3 and is not shown on this graph. B. Heatmap of normalised expression values of 21633 genes across the 6 non reduced and 6 reduced (R) samples where green depicts high expression and red low expression values.

Mentions: To investigate if more genes could be identified after globin reduction we used the edgeR Bioconductor statistical R package to examine differential gene expression between the 6 globin reduced and 6 non-reduced samples. We identified 21,633 uniquely expressed genes in the 12 samples and 11,633 out of these 21,633 genes were detected at significantly higher levels (FDR 1%) in the globin reduced samples (Figure 2). There were 87 genes detected at significantly lower levels in the globin reduced samples. The transcripts that were the most significantly decreased (p-value) were the pseudogene CXorf25 and the 4 globin transcripts globin delta (HBD), hemoglobin beta (HBB), hemoglobin alpha 1(HBA1), hemoglobin alpha 2 (HBA2) (see Table 2). Furthermore, 82 other non-globin transcripts were significantly reduced by the globin reduction procedure (Additional File 3).


Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA.

Mastrokolias A, den Dunnen JT, van Ommen GB, 't Hoen PA, van Roon-Mom WM - BMC Genomics (2012)

Differential expression of non-globin reduced versus globin reduced whole blood samples. A. MA-plot (log concentration on x-axis, log fold-change on y-axis) showing that the majority of differentially expressed genes (P value < 0.01, indicated in red) are up-regulated while the 4 globin genes showed the strongest reduction. Genes with zero expression in one of the groups are shown on the left end of the plot. HBD: hemoglobin delta, HBB: hemoglobin beta, HBA1: hemoglobin alpha 1, HBA1: hemoglobin alpha 2. CXorf25 has a logConc of -18.79 and a logFC of -14.3 and is not shown on this graph. B. Heatmap of normalised expression values of 21633 genes across the 6 non reduced and 6 reduced (R) samples where green depicts high expression and red low expression values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275489&req=5

Figure 2: Differential expression of non-globin reduced versus globin reduced whole blood samples. A. MA-plot (log concentration on x-axis, log fold-change on y-axis) showing that the majority of differentially expressed genes (P value < 0.01, indicated in red) are up-regulated while the 4 globin genes showed the strongest reduction. Genes with zero expression in one of the groups are shown on the left end of the plot. HBD: hemoglobin delta, HBB: hemoglobin beta, HBA1: hemoglobin alpha 1, HBA1: hemoglobin alpha 2. CXorf25 has a logConc of -18.79 and a logFC of -14.3 and is not shown on this graph. B. Heatmap of normalised expression values of 21633 genes across the 6 non reduced and 6 reduced (R) samples where green depicts high expression and red low expression values.
Mentions: To investigate if more genes could be identified after globin reduction we used the edgeR Bioconductor statistical R package to examine differential gene expression between the 6 globin reduced and 6 non-reduced samples. We identified 21,633 uniquely expressed genes in the 12 samples and 11,633 out of these 21,633 genes were detected at significantly higher levels (FDR 1%) in the globin reduced samples (Figure 2). There were 87 genes detected at significantly lower levels in the globin reduced samples. The transcripts that were the most significantly decreased (p-value) were the pseudogene CXorf25 and the 4 globin transcripts globin delta (HBD), hemoglobin beta (HBB), hemoglobin alpha 1(HBA1), hemoglobin alpha 2 (HBA2) (see Table 2). Furthermore, 82 other non-globin transcripts were significantly reduced by the globin reduction procedure (Additional File 3).

Bottom Line: Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples.In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples.The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Human and Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, 2333ZC, Leiden, The Netherlands.

ABSTRACT

Background: Transcriptome analysis is of great interest in clinical research, where significant differences between individuals can be translated into biomarkers of disease. Although next generation sequencing provides robust, comparable and highly informative expression profiling data, with several million of tags per blood sample, reticulocyte globin transcripts can constitute up to 76% of total mRNA compromising the detection of low abundant transcripts. We have removed globin transcripts from 6 human whole blood RNA samples with a human globin reduction kit and compared them with the same non-reduced samples using deep Serial Analysis of Gene Expression.

Results: Globin tags comprised 52-76% of total tags in our samples. Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples. In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples. Removing globin transcripts allowed us to also identify 2112 genes that could not be detected in the non-globin reduced samples, with roles in cell surface receptor signal transduction, G-protein coupled receptor protein signalling pathways and neurological processes.

Conclusions: The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

Show MeSH
Related in: MedlinePlus