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Endogenous plasma resuspension of peripheral blood mononuclear cells prevents preparative-associated stress that modifies polyA-enriched RNA responses to subsequent acute stressors. | LitMetric

AI Article Synopsis

  • Human peripheral blood mononuclear cells (PBMCs) were studied to understand biological processes and diseases, revealing significant variability in cellular activation and splicing.
  • The use of poly-A enriched RNA-sequencing identified over 42,000 gene expressions, including nearly all top genes from the Genotype Tissue Expression Project and some clinically-relevant genes.
  • The study found that preparing PBMCs in plasma preserved their characteristics better than conventional methods, leading to more consistent results when investigating acute cellular responses, emphasizing the importance of preparation techniques in reducing experimental variability.

Article Abstract

Human peripheral blood mononuclear cells (PBMCs) are used to examine biological processes and disease, when basal variability in cellular activation and splicing is described and unexplained. Using isolation systems that maintained buffy coat cells (PBMCs, platelets) in their own plasma, poly-A enriched RNA-sequencing (RNASeq) detected 42,720 Ensembl gene IDs, including >95% of the top 100 Genotype Tissue Expression Project (GTEx)-expressed genes in lung, colon, heart, skeletal muscle and liver, and 10/17 clinically-actionable genes listed by the Pharmacogenomics Knowledgebase. Transcriptome changes were defined after 1h treatment with 32°C hypothermia (hsp70 family member change), 10 μmol/L ferric citrate that had no discernible effect, and 100 μg/mL cycloheximide leading to induction of primary response (immediate early) genes including IL1B and TNF. Same-donor PBMCs prepared conventionally using washes then resuspension in serum-supplemented media demonstrated basal upregulation of stress signalling pathway genes that masked and overlapped differential gene expression profiles after 100 µg/L cycloheximide. Plasma-resuspended PBMCs demonstrated minor transcriptome changes after 40 μmol/L ferric citrate, whereas consistent and greater magnitude changes were observed for washed/media-resuspended PBMCs. We conclude that endogenous plasma-maintained PBMCs provide a more robust platform to interrogate acute cellular perturbations triggering innate immunity, and that varying susceptibility of PBMCs to preparative stresses is an important component of experimental variability.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11613960PMC
http://dx.doi.org/10.15698/cst2024.11.301DOI Listing

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