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

Abstract

Preserving the in vivo cell transcriptome is essential for accurate profiling, yet factors during cell isolation including time ex vivo and temperature induce artifactual gene expression, particularly in stress-responsive immune cells. In this study, we investigated two methods to mitigate ex vivo activation 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 subsequent in vitro experiments. 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. HighlightsO_LITraditional room temperature isolation methods trigger activation signature gene expression in PBMCs, even when rapidly isolated, whereas 4{degrees}C isolation methods do not. C_LIO_LITranscription and translation inhibitors and cold processing techniques reduce activation signature gene expression via shared mechanisms. C_LIO_LIPBMCs treated with transcription and translation inhibitors lose responsiveness to external stimuli. C_LIO_LICold isolation methods offer a suitable and inexpensive alternative to mitigate activation signature gene expression in PBMCs. C_LI