Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE33701 | GEO | 2012/01/25
SECONDARY ACCESSION(S): PRJNA148285
REPOSITORIES: GEO
Similar Datasets
Project description: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. Comparison of 6 whole blood RNA samples versus their globin reduced counterparts
2012-01-25 | E-GEOD-33701 | biostudies-arrayexpress
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 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.
2009-06-20 | E-GEOD-16728 | biostudies-arrayexpress
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
2009-06-20 | GSE16728 | GEO
Project description:In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. We compared technical and biological replicates having undergone globin depletion or not and found that globin depletion removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Peripheral whole blood transcriptome assessed by RNA-Seq on Illumina HiSeq 2000 in 6 healthy individuals and 6 pooled samples, either globin depleted or not.
2014-02-27 | E-GEOD-53655 | biostudies-arrayexpress
Project description:In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. We compared technical and biological replicates having undergone globin depletion or not and found that globin depletion removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures.
2014-02-27 | GSE53655 | GEO
Project description:A highly selective and non-genotoxic G9a inhibitor, RK-701 was discovered, which upregulated the mRNA level of γ-globin but not β-globin both in human erythroid cells and in mice. Using RK-701, we examined the induction of the fetal (γ-) globin protein in human erythroid cells; HUDEP-2 and human CD34+ bone marrow and peripheral blood cells.
2023-03-11 | PXD033536 | Pride
Project description:Background: There are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and rRNA are frequent contaminants. Given the abundance of erythrocytes in whole blood, globin genes comprise some 80% or more of the total RNA. Therefore, depletion of globin gene RNA and rRNA are critical steps required to have adequate coverage of reads mapping to the reference transcripts and thus reduce the total cost of sequencing. In this study, we directly compared the performance of probe hybridization (GLOBINClear Kit and Globin-Zero Gold rRNA Removal Kit) and RNAse-H enzymatic depletion (NEBNext® Globin & rRNA Depletion Kit and Ribo-Zero Plus rRNA Depletion Kit) methods from 1 ug of whole blood-derived RNA on mRNA-Seq profiling. All RNA samples were treated DNaseI for additional cleanup before the depletion step and were processed for poly-A selection for library generation. Results: Probe hybridization revealed a better overall performance than the RNAse-H enzymatic depletion method, detecting a higher number of genes and transcripts without 3’ region bias. After depletion, samples treated with probe hybridization showed globin genes at 0.5% (±0.6%) of the total mapped reads; the RNAse-H enzymatic depletion had 3.2% (±3.8%). Probe hybridization showed more junction reads and transcripts compared with RNAse-H enzymatic depletion and also had a higher correlation (R>0.9) than RNAse-H enzymatic depletion (R>0.85). Conclusion: In this study, our results showed that 1 µg of high-quality RNA from whole blood could be routinely used for transcriptional profiling analysis studies with globin gene and rRNA depletion pre-processing. We also demonstrated that the probe hybridization depletion method is better suited to mRNA sequencing analysis with minimal effect on RNA quality during depletion procedures.
2020-11-30 | GSE150587 | GEO
Project description:Peripheral whole blood-based gene expression profiling has become one of the most common strategies exploited in the development of clinically relevant biomarkers. However, the ability to identify biologically meaningful conclusions from gene expression patterns in whole blood is highly problematic. First, it is difficult to know whether or not expression patterns in whole blood capture those in primary tissues. Second, if explicit steps are not taken to accommodate the extremely elevated expression levels of globin in blood then large-scale multi-probe microarray-based studies can be severely compromised. Many studies consider the use of mouse blood as a model for human blood in addition to considering blood gene expression levels as a general surrogate for gene expression levels in other tissues. We explored the effects of globin reduction on peripheral mouse blood in the detection of genes known to be expressed in human tissues. Globin reduction resulted in 1.) a significant increase in the number of probes detected (5840 ± 944 vs 12411 ± 1904); 2.) increased expression for 4128 probe sets compared to non-globin reduced blood (p < .001, ≥ two-fold); 3.) improved detection of genes associated with many biological pathways and diseases; and 4.) an increased ability to detect genes expressed in 27 human tissues (p < 10-4). This study suggests that although microarray-based mouse blood gene expression studies that do not consider the effects of globin are severely compromised, globin-reduced mouse whole blood gene expression studies can be used to capture the expression profiles of genes known to contribute to various human diseases. Whole and GLOBINclear-treated paired blood samples from 18 mice representing 3 mice from 6 different isogenic mice strains.
2010-07-22 | E-GEOD-19282 | biostudies-arrayexpress
Project description:Whole blood rather than purified peripheral blood mononuclear cells is likely to become the prime tissue using expression microarrays for disease predication or prognosis however excess of globin mRNA may reduce probe detection sensitivity. In our study, we assessed whether whole-blood or globin-reduced RNA gives the most robust and sensitive results to detect small gene expression changes in response to hormone replacement therapy exposure. Each sample (N = 12) were hybridized according to 3 different protocols: no globin reduction (controls), globin reduction using peptid nucleic acids (PNA) and using magnetic beads in the GlobinClear kit from Ambion. Finally, 7 and 4 technical replicates were conducted in no globin reduction and PNA groups, respectively. Both globin reduction approaches were mostly efficient at reducing globin RNA from cRNA. Samples processed by GlobinClear kit gave a very distinct gene expression profiles from the controls while samples processed with PNA gave an intermediary profile closest to the non globin reduction group with a slight increased sensitivity of transcript detection but a loss of reproducibility. Overall, no sign of higher sensitivity in detection of gene expression changes followed by hormone exposure was observed after globin reduction which was therefore judged not beneficial. Keywords: Groups comparaison To assess effect of globin reduction protocols on gene expression from whole-blood, we selected 12 postmenopausal women (6 HRT users and 6 non-HRT users) who were not using other medication than HRT at the time of blood sampling and in order to cover a wide body mass index (BMI) range in both HRT and non HRT users. The samples were also selected to contain the highest concentration of total extracted RNA. Each sample was hybridized according to 3 different protocols: no globin reduction (controls), globin reduction using PNAs, globin reduction using magnetic beads in the GlobinClear™ kit from Ambion. The reproducibility of the globin reduction method compared to the non globin reduction approach was investigated for the PNA method only since this method was the most effective and specific. We planned to conduct 7 technical replicates in each group (i.e. no globin reduction and PNA groups). During amplification process with PNA, 4 samples failed to reverse transcript long fragment of mRNA certainly due to inhibitory contamination and these arrays were therefore excluded from our analyses. One sample (sample 34) was amplified with PNA twice the same day to study technical reproducibility without amplification date effect. Finally, a total of 47 arrays were conducted including 7 and 4 technical replicates in no globin reduction and PNA group, respectively.
2008-02-11 | E-GEOD-7008 | biostudies-arrayexpress
Project description:Peripheral whole blood-based gene expression profiling has become one of the most common strategies exploited in the development of clinically relevant biomarkers. However, the ability to identify biologically meaningful conclusions from gene expression patterns in whole blood is highly problematic. First, it is difficult to know whether or not expression patterns in whole blood capture those in primary tissues. Second, if explicit steps are not taken to accommodate the extremely elevated expression levels of globin in blood then large-scale multi-probe microarray-based studies can be severely compromised. Many studies consider the use of mouse blood as a model for human blood in addition to considering blood gene expression levels as a general surrogate for gene expression levels in other tissues. We explored the effects of globin reduction on peripheral mouse blood in the detection of genes known to be expressed in human tissues. Globin reduction resulted in 1.) a significant increase in the number of probes detected (5840 ± 944 vs 12411 ± 1904); 2.) increased expression for 4128 probe sets compared to non-globin reduced blood (p < .001, ≥ two-fold); 3.) improved detection of genes associated with many biological pathways and diseases; and 4.) an increased ability to detect genes expressed in 27 human tissues (p < 10-4). This study suggests that although microarray-based mouse blood gene expression studies that do not consider the effects of globin are severely compromised, globin-reduced mouse whole blood gene expression studies can be used to capture the expression profiles of genes known to contribute to various human diseases.
2010-07-22 | GSE19282 | GEO