Impact of storage conditions on peripheral leukocytes transcriptome

Leukocytes reflect the physiological and pathological states of each individual, and transcriptomic data of leukocytes have been used to reflect health conditions. Since the overall impact of ex vivo conditions on the leukocyte transcriptome before RNA stabilization remains unclear, we evaluated the influence of temporary storage conditions on the leukocyte transcriptome through RNA sequencing. We collected peripheral blood with EDTA tubes, which were processed immediately or stored either at 4 °C or room temperature (RT, 18-22 °C) for 2 h, 6 h and 24 h. Total cellular RNA was extracted from 42 leukocyte samples after red blood cells lysis for subsequent RNA sequencing. We applied weighted gene co-expression network analysis to construct co-expression networks of mRNA and lncRNA among the samples, and then performed gene ontology (GO) term enrichment to explore possible biological processes affected by storage conditions. Storage conditions change the gene expression of peripheral leukocytes. Comparing with fresh leukocytes, storage for 24 h at 4 °C and RT affected 1515 (1.51%) and 10,823 (10.82%) genes, respectively. Pathway enrichment analysis identified nucleosome assembly enriched in up-regulated genes at both conditions. When blood was stored at RT for 24 h, genes involved in apoptotic signaling pathway, negative regulation of cell cycle and lymphocyte activation were upregulated, while the relative proportion of neutrophils was significantly decreased. Temporary storage conditions profoundly affect the gene expression profiles of leukocytes and might further change cell viability and state. Storage of blood samples at 4 °C within 6 h largely maintains their original transcriptome.