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Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube and standard density gradient

ABSTRACT: High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. In this study, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.

ORGANISM(S): Homo sapiens

PROVIDER: GSE67321 | GEO | 2015/08/28

SECONDARY ACCESSION(S): PRJNA279506

REPOSITORIES: GEO

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Project description: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.

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Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube and standard density gradient

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