Effect of storage conditions on peripheral leukocytes transcriptome

Abstract

IntroductionPeripheral blood leukocytes are essential components of the innate and adaptive immune responses. Their transcriptome reflects an individuals physiological and pathological state, consequently bulk and single-cell RNA sequencing have been used to assess health and disease. As RNA is dynamic and could be affected by ex vivo conditions before RNA stabilization, we have assessed the influence of temporary storage on the transcriptome. MethodsWe collected peripheral blood from six healthy donors and processed it immediately or stored it either at 4{square} or room temperature (RT, 18-22{square}) for 2h, 6h and 24h. Total cellular RNA was extracted from leukocytes after red blood cells lysis and the transcriptome analyzed using RNA sequencing. ResultsWe identified 152 up-regulated and 12 down-regulated coding genes in samples stored at 4{square} for 24h with most of the up-regulated genes related to nucleosome assembly. More coding genes changed expression at RT, with 1218 increased and 1480 decreased. The increased genes were particularly related to mRNA processing and apoptosis, while the decreased genes were associated with neutrophil activation and cytokine production, implying a reduced proportion of neutrophils, which was confirmed by leukocyte subsets analysis. Most house-keeping genes changed expression during storage, but genes such as TUBB and C1orf43 were relatively stable and could serve as reference genes. ConclusionTemporary storage conditions profoundly affect leukocyte gene expression profiles and change leukocyte subset proportions. Blood samples stored at 4{square} for 6h largely maintain their original transcriptome.