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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

Transcript abundance in the non-globin reduced samples. Pie chart showing the most abundant transcripts in whole blood (average of 6 samples). Hemoglobin alpha 1 (HBA1), hemoglobin beta (HBB) and hemoglobin alpha 2 (HBA2) comprise the majority of transcripts.
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Figure 1: Transcript abundance in the non-globin reduced samples. Pie chart showing the most abundant transcripts in whole blood (average of 6 samples). Hemoglobin alpha 1 (HBA1), hemoglobin beta (HBB) and hemoglobin alpha 2 (HBA2) comprise the majority of transcripts.

Mentions: To investigate whether the RNA quality was affected by globin reduction, the RNA Integrity Number (RIN) value was determined for each sample (summarized in Table 1) with a RIN value of 10 representing the highest RNA quality. On average, the RIN value was slightly lower in the globin-reduced samples compared to the non-reduced samples indicating a minor reduction in RNA quality. Additionally, we measured RNA concentrations before and after globin reduction and observed an RNA yield loss of 5-9%. General sequencing and alignment statistics are given in Table 1. To verify if globin reduction was successful, the percentage of globin transcripts was determined in the reduced and non-reduced samples. The most abundant transcripts in the non-reduced samples corresponded to globin alpha1 (HBA1), globin beta (HBB) and globin alpha2 (HBA2) (Figure 1). The percentage of globin transcripts constituted on average 60% (52-76%) of total reported alignments and for the reduced samples this dropped to 0.1% - 0.4% (Table 1), demonstrating the efficiency of the globin reduction protocol. The two transcripts that were most abundant after the globin transcripts, lysozyme (LYZ) and ribosomal protein (RPLP2) transcripts were detected at higher levels in the globin-reduced samples. In order to validate the next generation sequencing results we performed qPCR for HBA and HBB for 4 non reduced and reduced samples and the results confirmed the next generation sequencing results (see Additional file 1).


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)

Transcript abundance in the non-globin reduced samples. Pie chart showing the most abundant transcripts in whole blood (average of 6 samples). Hemoglobin alpha 1 (HBA1), hemoglobin beta (HBB) and hemoglobin alpha 2 (HBA2) comprise the majority of transcripts.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Transcript abundance in the non-globin reduced samples. Pie chart showing the most abundant transcripts in whole blood (average of 6 samples). Hemoglobin alpha 1 (HBA1), hemoglobin beta (HBB) and hemoglobin alpha 2 (HBA2) comprise the majority of transcripts.
Mentions: To investigate whether the RNA quality was affected by globin reduction, the RNA Integrity Number (RIN) value was determined for each sample (summarized in Table 1) with a RIN value of 10 representing the highest RNA quality. On average, the RIN value was slightly lower in the globin-reduced samples compared to the non-reduced samples indicating a minor reduction in RNA quality. Additionally, we measured RNA concentrations before and after globin reduction and observed an RNA yield loss of 5-9%. General sequencing and alignment statistics are given in Table 1. To verify if globin reduction was successful, the percentage of globin transcripts was determined in the reduced and non-reduced samples. The most abundant transcripts in the non-reduced samples corresponded to globin alpha1 (HBA1), globin beta (HBB) and globin alpha2 (HBA2) (Figure 1). The percentage of globin transcripts constituted on average 60% (52-76%) of total reported alignments and for the reduced samples this dropped to 0.1% - 0.4% (Table 1), demonstrating the efficiency of the globin reduction protocol. The two transcripts that were most abundant after the globin transcripts, lysozyme (LYZ) and ribosomal protein (RPLP2) transcripts were detected at higher levels in the globin-reduced samples. In order to validate the next generation sequencing results we performed qPCR for HBA and HBB for 4 non reduced and reduced samples and the results confirmed the next generation sequencing results (see Additional file 1).

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