(re)freezing PBMC

This study investigates the impact of cryopreservation and freezing media on RNA quality and gene expression profiles of peripheral blood mononuclear cells (PBMC). No significant difference in cell viability or RNA quality was observed between freshly isolated and cryopreserved cells, even after two freeze-thaw cycles. Transcriptome analysis revealed no significant differences in alignment rates or read counts across various conditions. Differential gene expression analysis identified changes between fresh and thawed samples, with a consistent downward trend of mRNA abundance in the frozen samples, but minimal changes were observed between the first and second freeze-thaw cycles. Gene set enrichment analysis showed only minor significant differences, and stress-related gene sets were not upregulated. Freezing medium appears to be the safer option compared to freezing in trizol. These results confirm that cryopreservation and thawing processes do not compromise RNA integrity or sequencing reliability in PBMC, supporting the use of cryopreserved samples for gene expression studies.