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Deciphering the Gene Expression Pattern from Human Blood Samples Collected in Multiple Collection Tubes

Theobjective of this study was to investigate how blood storage, extraction methodologies, and the actual blood component itself may influence what genes are expressed in downstream applications. To discover potential biases, blood was collected in triplicate from five donors into RNAgard Blood Tubes, PAXgene Blood RNA Tubes, EDTA Tubes, Sodium Citrate Cell Preparation Tubes (CPT), and Blood Collection Tubes with Acid Citrate Dextrose Solution A (ACD‐A). The RNA was isolated from whole blood and peripheral blood mononuclear cells (PBMCs) using ten different combinations of experimental conditions combined with several widelyutilized RNA isolation methods. After extraction, the RNA was quantified using a spectrophotometer and RNA quality was assessed by the RNA Integrity Number (RIN) value. The RNA was then profiled using Agilent Human Gene Expression micro arrays to observe the expression levels for the different experimental conditions. When the RNA quality was assessed by the RIN value, we found that all PBMC procedures had the highest RIN values as they were processed and stabilized in TRIzol Reagent prior to RNA extraction. Initial data analysis showed that human blood stored at 4°C overnight performed equally well when compared to frozen stabilized blood. We compared within and across donor and method replicates, which indicated signal to noise patterns that were not captured by RIN value alone. Our results provide some new insights into RNA isolation from blood samples and how it is influenced by the choice of storage, handling, and methodologies, and that careful consideration is necessary to avoid bias in downstream processing. Better characterization of collection method idiosyncrasies will facilitate further research in to understanding the effect of gene expression on variability in human sample types.