Investigating Gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple methods

Gene expression profiling using blood samples is a valuable tool for biomarker discovery in clinical studies. Different whole blood RNA collection and processing methods are highly variable and might confound comparisons of results across studies. The main aim of the study was to compare how blood storage, extraction methodologies, and the blood component itself may influence gene expression profiling where samples were collected in triplicate from five healthy donors. Whole blood was collected in RNAGard™, PAXgene™ as well as collection tubes with anticoagulant such as dipotassium ethylenediaminetetraacetic acid (K2EDTA) and acid citrate dextrose solution A (ACD-A). Peripheral blood mononuclear cells (PBMCs) were separated using sodium citrate cell preparation tubes (CPT), FICOLL™, immunomagnetic and Leukolock™ methods. The Leukolock™, K2EDTA and ACD-A blood tubes were shipped overnight using cold conditions and rest of the methods were immediately frozen with or without pre-processing. The RNA was isolated from whole blood and peripheral blood mononuclear cells (PBMCs) with a total of 10 different experimental conditions employing several widely utilized RNA isolation methods. The RNA quality as assessed by RNA integrity Number (RIN) showed that all PBMC procedures had the highest RIN values when processed and stabilized in TRIzol before RNA extraction. Initial data analysis showed that human blood stored at 4°C overnight performed equally well when compared to frozen stabilized blood. Comparisons within and across donor/method replicates showed signal to noise patterns which were not captured by RIN value alone. Our results provide some new insights into RNA isolation from blood samples, how the RNA isolation quality and apparent gene expression is influenced by the choice of storage, handling, and methodologies, and how careful consideration is necessary to avoid bias resulting from downstream processing. Better characterization of the effects of collection method idiosyncrasies will facilitate further research into understanding the effect of gene expression on variability in human sample types. The objective of this study was to investigate how blood storage, extraction methodologies, and the blood component itself may influence what genes appear to be expressed in downstream applications. Blood was collected in triplicate from 5 donors into five different collection tubes. Few of the tubes were frozen post collection, while others were stored at 4°C overnight. The RNA was isolated from whole blood and peripheral blood mononuclear cells (PBMCs) employing several widely utilized 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 microarrays to investigate the different expression levels for the experimental conditions. The RNA quality was assessed by RIN value and we found out that all PBMC procedures had the highest RIN values when processed and stabilized in TRIzol before RNA extraction. Initial data analysis showed that human blood stored at 4°C overnight performed equally well when compared to frozen stabilized blood. Comparisons of blood collection methods within and across donor replicates showed signal to noise patterns which were not captured by RIN value alone. Our results provide some new insights into RNA isolation from blood samples, how the RNA isolation quality and apparent gene expression is influenced by the choice of storage, handling, and methodologies, and how careful consideration is necessary to avoid bias resulting from downstream processing. Better characterization of the effects of collection method idiosyncrasies will facilitate further research into understanding the effect of gene expression on variability in human sample types. [Collection protocol] Blood was collected and preserved in five tubes, namely, PAXgene™ RNA tubes, RNAgard® Blood Tubes, BD Vacutainer™ tubes with ACD-A and K2EDTA and CPT (LeukoLOCK) tubes from five healthy volunteers. In parallel, PBMCs were isolated from EDTA, CPT and ACD-A tubes following the manufacturer’s instructions.