title: The effect of cell subset isolation method on gene expression in leukocytes.
abstract: Multiple scientific disciplines require the isolation of specific subsets of blood cells from patient samples for gene expression analysis by microarray or RNA-sequencing, preserving disease- or treatment-related signatures. However, little is known with respect to the impact of different cell isolation methods on gene expression and the effects of positive selection, negative selection, and fluorescence activated cell sorting (FACS) have not previously been assessed in parallel. To address this knowledge gap, CD4+ T cells, CD8+ T cells, B cells, and monocytes were isolated from blood samples from five independent donors using positive immunomagnetic selection, negative immunomagnetic selection, and FACS. We hypothesized that positive selection and FACS would yield higher purity but may have an impact on gene expression since both methods utilize antibodies that bind surface receptors of the cell type of interest. Moreover, FACS might upregulate stress response genes due to passage of the cells through the sorter. Microarray gene expression data were generated and subjected to unsupervised clustering and differential gene expression analysis. Surprisingly, these analyses revealed that gene expression signatures were more similar between cells isolated by negative selection and FACS compared to cells isolated by positive selection. Moreover, genes that are involved in the response to stress generally had the highest expression in cells isolated by negative or positive selection and not FACS. Thus, FACS is the recommended method for isolation of leukocyte subsets for gene expression studies since this method results in the purest subset populations and does not appear to induce a stress response.
authors.affiliation: Department of Medicine, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Nadejda
authors.initials: N
authors.lastname: Beliakova-Bethell
authors.affiliation: Institut de recerca de la Sida-IrsiCaixa, Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol, IGTP, Badalona, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Massanella
authors.affiliation: Department of Medicine, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Cory
authors.initials: C
authors.lastname: White
authors.affiliation: Veterans Affairs San Diego Healthcare System, San Diego, California 92161, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Lada
authors.affiliation: Veterans Affairs San Diego Healthcare System, San Diego, California 92161, USA.
authors.forename: Pinyi
authors.initials: P
authors.lastname: Du
authors.affiliation: Department of Family and Preventive Medicine, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Florin
authors.initials: F
authors.lastname: Vaida
authors.affiliation: Institut de recerca de la Sida-IrsiCaixa, Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol, IGTP, Badalona, Spain.
authors.forename: Julià
authors.initials: J
authors.lastname: Blanco
authors.affiliation: Veterans Affairs San Diego Healthcare System, San Diego, California 92161, USA.|Department of Pathology, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Celsa A
authors.initials: CA
authors.lastname: Spina
authors.affiliation: Department of Medicine, University of California San Diego, La Jolla, California 92093, USA.|Veterans Affairs San Diego Healthcare System, San Diego, California 92161, USA.|Faculty of Medicine, University of Southampton, Southampton, Hants SO16 6YD, UK.
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Woelk
mesh_terms: D001402:B-Lymphocytes; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D007962:Leukocytes / Q000145:classification / Q000166:cytology*; D009000:Monocytes / Q000166:cytology
keywords: B cell; CD4+ T cell; CD8+ T cell; fluorescent activated cell sorting; gene expression; microarray; monocyte; negative immunomagnetic selection; positive immunomagnetic selection