The effect of cell isolation methods on the human transcriptome profiling and microbial transcripts of peripheral blood.

The expression of human and microbial genes serves as biomarkers for disease and health. Blood RNA is an important biological resource for precision medicine and translational medicine. However, few studies have assessed the human transcriptome profiles and microbial communities composition and diversity of peripheral blood from different cell isolation methods, which could affect the reproducibility of researches. We collected peripheral blood from three healthy donors and processed it immediately. We used RNA sequencing to investigate the effect of three leukocyte isolation methods including buffy coat (BC) extraction, red blood cell (RBC) lysis and peripheral blood mononuclear cell (PBMC) isolation with the comparison with whole blood (WB), through analyzing the sensitivity of gene detection, the whole transcriptome profiling and microbial composition and diversity. Our data showed that BC extraction with high globin mRNA mapping rate had similar transcriptome profiles with WB, while RBC lysis and PBMC isolation depleted RBCs effectively. With the efficient depletion of RBC and distinct compositions of leukocyte subsets, RNA-seq of RBC lysis and PBMC isolation uniquely detected genes from specific cell types, like granulocytes and NK cells. In addition, we observed that the microbial composition and diversity were more affected by individuals than isolation methods. Our results showed that blood cell isolations could largely influence the sensitivity of detection of human genes and transcriptome profile.

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.