Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE53655 | GEO | 2014/02/27
SECONDARY ACCESSION(S): PRJNA232593
REPOSITORIES: GEO
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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: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: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: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
2008-02-12 | GSE7008 | GEO
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:Background: RNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript. Results: Independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage. Conclusions: Technical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases.
2014-01-15 | GSE54068 | GEO
Project description:Background: RNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript. Results: Independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage. Conclusions: Technical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases.
2014-01-15 | GSE54067 | GEO
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:We extracted RNA from C57BL/6 mouse neutrophils to measure inter-individual variability in gene expression. 5 of the 10 blood samples were analyzed separately, the other 5 were pooled then split into 5 technical replicates to measure technical variability. We could then measure inter-individual variability in gene expression and compare it to the amplitude of miRNA-guided repression by miR-223.
2016-09-21 | GSE59549 | GEO
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