Figure 8. The fold change in gene expression after LPS- stimulation predicted in PBMC as a function of fold change in monocytes. The top curve is for genes expressed only in monocytes with no attenuation of fold change. The other curves assume that the gene is expressed in monocytes at double, equal and half the level that it is in non-monocytes. Expression of the IL-1a gene at 3 and 24 hours is plotted as an example. doi:10.1371/journal.pone.0004427.g008 incubated in phosphate buffered saline (PBS), 0.1% sodium azide and 20 ml of the conjugated antibody at room temperature for 15 min, washed and resuspended in 300 ml PBS with 2% formalin. Analysis was undertaken using a LSR II flow cytometer and BD FACSDivaH software (Becton Dickinson). The Mono+ samples were not measured directly although selection with anti-CD14 Gene Expression & Cell Subsets beads is likely to select CD14-positive cells at an efficiency approaching 100%. This contention is supported by the MonoD sample having almost 0% CD14-positive cells. RNA preparation RNA was isolated by chloroform:phenol extraction[17] and purified using a kit (RNeasyTM, Qiagen, Clifton Hill, Victoria, Australia) following the manufacturer’s protocol. Linear amplifi- cation of RNA was performed using a kit (MessageAmpTM II aRNA, Ambion, Austin, TX, USA) following the manufacturer’s protocol. RNA quality was assessed by A260/A280 ratio in addition to analysis in a Bioanalyzer 2100 (Agilent Technologies, Forest Hill, Victoria, Australia). All RNA samples were of a satisfactory quality. Microarray hybridisation The study used 36 spotted microarrays printed with the Compugen human 19,000 oligonucleotide library (http://www. cgen.com) and a selection of control probes at the Adelaide Microarray Facility (Adelaide, Australia). To minimize variability, all microarrays were from the same printing batch. Amplified RNA (aRNA) was labelled by a direct platinum-based labelling technique using a kit (ULSTM aRNA labelling, Kreatech Biotechnology, Amsterdam, The Netherlands) following the manufacturer’s protocol. For each sample 2 mg aRNA was labelled with 2 ml of ULSTM-Cy dye. For each pair of aRNA samples to be hybridised to a slide, one sample was labelled with ULSTM-Cy3 and one with ULSTM-Cy5. aRNA from each stimulation time point was competitively hybridised with aRNA from 0 hours of the same cell type from the same individual. The samples from each individual were hybridised separately. The two dye-coupled samples for each array were combined and fragmented using 4 ml Fragmentation Reagents (Ambion). The labelled sample was mixed with 10 ml 1 mg/ml human Cot-1 DNA (Invitrogen), 15 ml 206SSC, 20 ml deionised formamide (Sigma Aldrich), 20 ml Kreatech solution (Kreatech Biotechnolo- Figure 9. Examples of genes that are expressed in opposite directions in monocytes and PBMC after stimulation with LPS for 3 hours. The log2-fold change (M value) and standard error for the expression of each gene are plotted for each sample. doi:10.1371/journal.pone.0004427.g009 PLoS ONE | www.plosone.org 9 February 2009 | Volume 4 | Issue 2 | e4427