Comparative Analysis of Methods to Reduce Activation Signature Gene Expression in PBMCs

Abstract

SummaryPreserving thein vivocell transcriptome is essential for accurate profiling, yet factors during cell isolation including timeex vivoand temperature induce artifactual gene expression, particularly in stress-responsive immune cells. In this study, we investigated two methods to mitigateex vivoactivation signature gene (ASG) expression in peripheral blood mononuclear cells (PBMCs): transcription and translation inhibitors (TTis) and cold temperatures during isolation. Comparative analysis of PBMCs isolated with TTis revealed reduced ASG expression. However, TTi treatment impaired responsiveness to LPS stimulation in subsequentin vitroexperiments. In contrast, cold isolation methods also prevented ASG expression; up to a point where the addition of TTis during cold isolation offered minimal additional advantage. These findings highlight the importance of considering the advantages and drawbacks of different isolation methods to ensure accurate interpretation of PBMC transcriptomic profiles.HighlightsTraditional room temperature isolation methods trigger activation signature gene expression in PBMCs, even when rapidly isolated, whereas 4°C isolation methods do not.Transcription and translation inhibitors and cold processing techniques reduce activation signature gene expression via shared mechanisms.PBMCs treated with transcription and translation inhibitors lose responsiveness to external stimuli.Cold isolation methods offer a suitable and inexpensive alternative to mitigate activation signature gene expression in PBMCs.