Abstract
BackgroundReadily accessible samples such as peripheral blood or cell lines are increasingly being used in large cohorts to characterise gene expression differences between a patient group and healthy controls. However, cell and RNA isolation procedures and the variety of cell types that make up whole blood can affect gene expression measurements. We therefore systematically investigated global gene expression profiles in peripheral blood from six individuals collected during two visits by comparing five of the following cell and RNA isolation methods: whole blood (PAXgene), peripheral blood mononuclear cells (PBMCs), lymphoblastoid cell lines (LCLs), CD19 and CD20 specific B-cell subsets.ResultsGene expression measurements were clearly discriminated by isolation method although the reproducibility was high for all methods (range ρ = 0.90-1.00). The PAXgene samples showed a decrease in the number of expressed genes (P < 1*10-16) with higher variability (P < 1*10-16) compared to the other methods. Differentially expressed probes between PAXgene and PBMCs were correlated with the number of monocytes, lymphocytes, neutrophils or erythrocytes. The correlations (ρ = 0.83; ρ = 0.79) of the expression levels of detected probes between LCLs and B-cell subsets were much lower compared to the two B-cell isolation methods (ρ = 0.98). Gene ontology analysis of detected genes showed that genes involved in inflammatory responses are enriched in B-cells CD19 and CD20 whereas genes involved in alcohol metabolic process and the cell cycle were enriched in LCLs.ConclusionGene expression profiles in blood-based samples are strongly dependent on the predominant constituent cell type(s) and RNA isolation method. It is crucial to understand the differences and variability of gene expression measurements between cell and RNA isolation procedures, and their relevance to disease processes, before application in large clinical studies.
Highlights
Accessible samples such as peripheral blood or cell lines are increasingly being used in large cohorts to characterise gene expression differences between a patient group and healthy controls
We investigated variability and consistency in gene expression profiles between five of the most common post venipuncture methods of cell and RNA isolation: whole blood (PAXgene (PAX)), peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs), CD19-specific B-cells subsets (B-cell CD19), CD20-specific B-cells subsets (B-cell CD20)
Using samples from six individuals collected during two visits, we evaluated the differences and concordances of global gene expression profiles, the biological and technical variability seen in these approaches, cell-type specific gene expression signatures and their relevance to large-scale biobanking initiatives
Summary
Accessible samples such as peripheral blood or cell lines are increasingly being used in large cohorts to characterise gene expression differences between a patient group and healthy controls. Alongside genome-wide association studies and upcoming sequencing studies, there is increasing interest in obtaining large-scale “omics” data from large biobanks and sample collections, including gene expression, proteomic and metabonomic profiling. These biobanks will rely on easy sample collection and handling using robust methodologies and sample storage over a prolonged time period. While the downstream gene expression profiling techniques using microarrays are very reliable for large-scale investigations, there are still challenges prior to microarray analysis including the choice of a relevant sample type and RNA and cell isolation method. Other isolation methods attempt to generate a subset of cell types such as peripheral blood mononuclear cells (PBMCs) by the use of Ficoll or lymphocyte subsets using magnetic beads
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