Single-cell RNA Sequencing data from PBMC subjected to whole blood processing delay
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PROVIDER: PRJNA672734 | ENA |
REPOSITORIES: ENA
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Project description:Single-cell RNA Sequencing data from PBMC subjected to whole blood processing delay
| PRJNA672733 | ENA
Project description:In the analysis of peripheral blood gene expression, timely processing of samples is essential to ensure that measurements reflect in vivo biology, rather than ex vivo sample processing variables. The effect of processing delays on global gene expression patterns in peripheral blood mononuclear cells (PBMC) was assessed by isolating and stabilizing PBMC-derived RNA from three individuals either immediately after phlebotomy or following a 4 hour delay. RNA was labeled using NuGEN Ovation labeling and probed using the Affymetrix HG U133plus 2.0 GeneChip®. Comparison of gene expression levels (p<0.05 and ? 2-fold expression change) identified 327 probe sets representing genes with increased expression and 46 indicating decreased expression after 4 hours. The trends in expression patterns associated with delayed processing were also apparent in an independent set of 276 arrays of RNA from human PBMC samples with varying processing times. These data indicate that the time between sample acquisition, initiation of processing, and when the RNA is stabilized should be a prime consideration when designing protocols for translational studies involving PBMC gene expression analysis. Keywords: Technical comparison 3 subjects, 3 samples each. First sample is prepared immediately, 2nd after 2 hour delay and last after a 4 hour delay.
2011-03-22 | E-GEOD-21039 | biostudies-arrayexpress
Project description:In the analysis of peripheral blood gene expression, timely processing of samples is essential to ensure that measurements reflect in vivo biology, rather than ex vivo sample processing variables. The effect of processing delays on global gene expression patterns in peripheral blood mononuclear cells (PBMC) was assessed by isolating and stabilizing PBMC-derived RNA from three individuals either immediately after phlebotomy or following a 4 hour delay. RNA was labeled using NuGEN Ovation labeling and probed using the Affymetrix HG U133plus 2.0 GeneChip®. Comparison of gene expression levels (p<0.05 and ≥ 2-fold expression change) identified 327 probe sets representing genes with increased expression and 46 indicating decreased expression after 4 hours. The trends in expression patterns associated with delayed processing were also apparent in an independent set of 276 arrays of RNA from human PBMC samples with varying processing times. These data indicate that the time between sample acquisition, initiation of processing, and when the RNA is stabilized should be a prime consideration when designing protocols for translational studies involving PBMC gene expression analysis. Keywords: Technical comparison
2011-03-22 | GSE21039 | GEO
Project description:BackgroundIn studies of perfluoroalkyl acids, the validity and comparability of measured concentrations may be affected by differences in the handling of biospecimens. We aimed to investigate whether measured plasma levels of perfluoroalkyl acids differed between blood samples subjected to delay and transportation prior to processing and samples with immediate processing and freezing.MethodsPregnant women recruited at Aarhus University Hospital, Denmark, (n = 88) provided paired blood samples. For each pair of samples, one was immediately processed and plasma was frozen, and the other was delayed and transported as whole blood before processing and freezing of plasma (similar to the Danish National Birth Cohort). We measured 12 perfluoroalkyl acids and present results for compounds with more than 50% of samples above the lower limit of quantification.ResultsFor samples taken in the winter, relative differences between the paired samples ranged between -77 and +38% for individual perfluoroalkyl acids. In most cases concentrations were lower in the delayed and transported samples, e.g. the relative difference was -29% (95% confidence interval -30; -27) for perfluorooctane sulfonate. For perfluorooctanoate there was no difference between the two setups [corresponding estimate 1% (0, 3)]. Differences were negligible in the summer for all compounds.ConclusionsTransport of blood samples and processing delay, similar to conditions applied in some large, population-based studies, may affect measured perfluoroalkyl acid concentrations, mainly when outdoor temperatures are low. Attention to processing conditions is needed in studies of perfluoroalkyl acid exposure in humans.
| S-EPMC4565678 | biostudies-literature
Project description:PBMC Fixation and Processing for Chromium Single-Cell RNA Sequencing
| PRJNA449292 | ENA
Project description:Human peripheral blood mononuclear cells (PBMC) are key to several diagnostics assays and basic science research. Blood pre-analytical variations that occur before obtaining the PBMC fraction can have a significant impact on the results of the assays, including viability, composition, integrity, and gene expression changes of immune cells. With this as motivation, we performed a quantitative shotgun proteomics analysis using Isobaric Tag for Relative and Absolute Quantitation (iTRAQ 8plex) labeling to compare PBMC obtained from fresh versus 24h-stored blood at room temperature. We identified 3,195 proteins, of which 245 were differentially abundant. Our results revealed enriched pathways in the fresh-PBMC samples related to exocytosis, localization, vesicle-mediated transport, cell activation, and secretion. In contrast, pathways related to exocytosis, neutrophil degranulation and activation, granulocyte activation, leukocyte degranulation, and myeloid leukocyte activation involved in immune response were enriched in the 24h-PBMC samples, which may indicate probable granulocyte contamination and activation due to blood storage time and temperature. Examples of upregulated proteins in the 24h-PBMC sample are: CAMP, S100A8, LTA4H, RASAL3, and S100A6, which are involved in an adaptive immune system and antimicrobial activity, proinflammatory mediation, aminopeptidase activities, and naïve T cells survival. Moreover, examples of downregulated proteins are: NDUFA5, TAGLN2, H3C1, TUBA8, and CCT2 that are related to the cytoskeleton, cell junction, mitochondrial respiratory chain. In conclusion, blood-processing time directly impacts the proteomic profile of human PBMC.
2020-12-14 | PXD021367 | Pride
Project description:The gene expression profiles of chicken whole blood (WB) and peripheral blood mononuclear cells (PBMC) have been characterized and compared by microarray analysis. This study highlights the respective advantages of each blood cell subset and show that whole blood is more suitable for large scale studies and allows for the exploration of biological processes which are so far poorly investigated. Whole blood and PBMC from four male broilers (9 weeks old) were collected and isolated before -80°C conservation.
2016-07-22 | E-GEOD-65331 | biostudies-arrayexpress
Project description:This dataset contains raw mass spectrometry data (in the form of Thermo Fisher .RAW files) supporting associated publication “Variable blood processing procedures contribute to plasma proteomic variability” by Halvey et al.
The experiments characterized by these data evaluated effects on human plasma proteome of the following factors involved in blood to plasma processing: delay to the first centrifugation, temperature and type of anticoagulant tube. Each combination of these factors has been applied to samples from n=4 healthy volunteers. Sample annotation provided in the file “Blood_to_Plasma_Processing_Sample_Annotation.txt” maps each sample to combination of those factors and anonymous subject identifier.
For “Experiment5” there were several instances of LC-MS/MS analytical replicates for this sample. It should be noted that for this experiment the .RAW file from the “./updates/2020-08-03_vfarutin_e264fbfc/raw/” folder under this dataset FTP address represents the actual LC-MS/MS run used to generate the data in the associated paper by Halvey et al.
2020-07-23 | MSV000085814 | MassIVE
Project description:The gene expression profiles of chicken whole blood (WB) and peripheral blood mononuclear cells (PBMC) have been characterized and compared by microarray analysis. This study highlights the respective advantages of each blood cell subset and show that whole blood is more suitable for large scale studies and allows for the exploration of biological processes which are so far poorly investigated.
2016-07-22 | GSE65331 | GEO
Project description:Background: Interest in single-cell whole transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works, investigators have used live cells which represent several inconveniences and limitations. Some recent cell fixation methods did not work with most primary cells including immune cells. Methods: The methanol-fixation and new processing method was introduced to preserve PBMCs for single-cell RNA sequencing (scRNA-Seq) analysis on 10X Chromium platform. Results: When methanol fixation protocol was broken up into three steps, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to three-month storage steps. Resuspension but not rehydration in 3X saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3X SSC were successfully implemented into 10X Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol was validated in CD8+ T cell and several other cell types. Conclusions: We expect that the methanol-based cell fixation procedure presented here will substantially enable complex experimental design with primary cells at single cell resolution.
2018-07-25 | GSE112845 | GEO