10Motivation: Copy-number variants (CNVs) are one of the major causes of genetic disorders. 11However, current methods for CNV calling have high false-positive rates and low concordance, and 12 a few of them can accurately genotype CNVs.

BACKGROUND: Plasma cell-free RNA (cfRNA) are potential biomarkers for disease prediction and diagnosis. However, pre-analysis factors, such as the delay in blood processing and storage may lead to unreliable results, though no study has systematically evaluated the effect of blood storage conditions on the whole transcriptome of plasma cfRNA yet. METHODS: We collected peripheral blood samples from four healthy subjects and allowed them to stand at room temperature or 4℃ for different time periods (0h, 2h, 6h and 24h) prior to plasma separation. Then, plasma cfRNA stability was evaluated by measuring expression changes of cell-free mRNA, lncRNA and miRNA using high throughput sequencing-based profiling. Finally, their paired leukocyte RNA data were integrated to depict the effect of leukocytes on plasma cfRNA during storage. RESULTS: Plasma mRNA and lncRNA presented high correlations (Pearson R2 ≥ 0.8) and fewer variations when blood was stored at 4℃ for 6 hours or stored at RT for 2 hours. miRNA was more stable, with minimal R2 of 0.86 at 4℃ for at least 24 hours or at RT for 6 hours. Correlations of plasma RNA and leukocyte RNA increased with the incubation time, and the relative proportion of neutrophils in plasma grown from 14.3% to 61.2% at RT (P = 0.004), indicating leukocyte RNA contamination. Besides, the tissue enriched genes in plasma were down-regulated with the extension of storage time. CONCLUSIONS: Our results characterized the effects of short-term storage of blood samples on plasma cfRNA, which will facilitate further researches or clinical applications to avoid bias resulting from sample processing.