Detection of Gene Expression in an Individual Cell Type within a Cell Mixture Using Microarray Analysis Penelope A. Bryant1,2,3, Gordon K. Smyth4, Roy Robins-Browne2,5, Nigel Curtis1,2,3* 1 Department of Paediatrics, The University of Melbourne, Royal Children’s Hospital Melbourne, Parkville, Victoria, Australia, 2 Microbiology & Infectious Diseases Research Group, Murdoch Children’s Research Institute, Royal Children’s Hospital Melbourne, Parkville, Victoria, Australia, 3 Infectious Diseases Unit, Department of General Medicine, Royal Children’s Hospital Melbourne, Parkville, Victoria, Australia, 4 Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, 5 Department of Microbiology & Immunology, University of Melbourne, Parkville, Victoria, Australia Abstract Background: A central issue in the design of microarray-based analysis of global gene expression is the choice between using cells of single type and a mixture of cells. This study quantified the proportion of lipopolysaccharide (LPS) induced differentially expressed monocyte genes that could be measured in peripheral blood mononuclear cells (PBMC), and determined the extent to which gene expression in the non-monocyte cell fraction diluted or obscured fold changes that could be detected in the cell mixture. Methodology/Principal Findings: Human PBMC were stimulated with LPS, and monocytes were then isolated by positive (Mono+) or negative (Mono2) selection. The non-monocyte cell fraction (MonoD) remaining after positive selection of monocytes was used to determine the effect of non-monocyte cells on overall expression. RNA from LPS-stimulated PBMC, Mono+, Mono2 and MonoD samples was co-hybridised with unstimulated RNA for each cell type on oligonucleotide microarrays. There was a positive correlation in gene expression between PBMC and both Mono+ (0.77) and Mono2 (0.61– 0.67) samples. Analysis of individual genes that were differentially expressed in Mono+ and Mono2 samples showed that the ability to detect expression of some genes was similar when analysing PBMC, but for others, differential expression was either not detected or changed in the opposite direction. As a result of the dilutional or obscuring effect of gene expression in non-monocyte cells, overall about half of the statistically significant LPS-induced changes in gene expression in monocytes were not detected in PBMC. However, 97% of genes with a four fold or greater change in expression in monocytes after LPS stimulation, and almost all (96–100%) of the top 100 most differentially expressed monocyte genes were detected in PBMC. Conclusions/Significance: The effect of non-responding cells in a mixture dilutes or obscures the detection of subtle changes in gene expression in an individual cell type. However, for studies in which only the most highly differentially expressed genes are of interest, separating and analysing individual cell types may be unnecessary. Citation: Bryant PA, Smyth GK, Robins-Browne R, Curtis N (2009) Detection of Gene Expression in an Individual Cell Type within a Cell Mixture Using Microarray Analysis. PLoS ONE 4(2): e4427. doi:10.1371/journal.pone.0004427 Editor: Sui Huang, University of Calgary, Canada Received July 17, 2008; Accepted December 12, 2008; Published February 12, 2009 Copyright: 2009 Bryant et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a grant from the National Health and Medical Research Council and National Heart Foundation of Australia. Dr. Bryant was the recipient of a European Society of Paediatric Infectious Diseases Fellowship Award and a University of Melbourne Research Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nigel.curtis@rch.org.au Introduction The choice between using cells of a single type and a mixture of cells in microarray-based analysis of global gene expression is difficult. Analysing cells of one type, such as monocytes[1,2,3], necessitates an isolation step that may be technically difficult[4] and which may induce non-specific changes in gene expression. Analysing a cell mixture, such as peripheral blood mononuclear cells (PBMC)[5,6,7], is simpler but gene expression changes in cells of a specific cell type, particularly if present in small numbers, may not be detected. Specifically, significant changes of gene expression in the cell type of interest may be undetectable as a result of the dilutional effect of a majority of non-responding cells or obscured by opposite responses in other cell types. In addition, it is impossible to attribute observed differential expression to individ- ual cell types. This study compared gene expression in PBMC with expression in monocytes alone after stimulation with lipopolysaccharide (LPS) as a paradigm for determining the extent to which changes in gene expression in a particular cell type can be detected in a cell mixture. A strategy was used that preserved the advantage of the cellular interactions of PBMC but which allowed detection of gene expression from an individual cell type by separating monocytes after stimulation of PBMC ex vivo. Analysis was undertaken in monocytes isolated by both positive and negative selection to control for possible gene expression induced by CD14-mediated pathways. Gene expression was also measured in the non- monocyte cell fraction remaining after positive selection to provide PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4427