Multimodal Analysis for Human ex vivo Studies Shows Extensive Molecular Changes from Delays in Blood Processing
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PROVIDER: PRJNA659732 | ENA |
REPOSITORIES: ENA
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Project description:Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMCs up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states.
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Project description:Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMC up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states.
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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.
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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
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