Figure 1. Schematic representation of the experimental design. See Materials and Methods for details. The key features of the design are the use of a single pair of RNA samples for all analyses, mixed together in varying proportions and analyzed in technical replicates on each platform. Unlike the Affymetrix plat- form, each Illumina BeadArray slide contains multiple arrays, allowing us to analyze a complete dilution series on one slide. Note that the BeadArray slides actually contain eight arrays per slide, but we only used six for the data described here. locations of each probe on the array are determined using a molecular address (1). A ﬁnal difference between the plat- forms is that in the current packaging, multiple Illumina arrays are placed on the same physical substrate, meaning that hybridization and other steps are performed in a parallel manner, while Affymetrix arrays are processed separately. This paper details results from an experiment comparing Affymetrix HG-U133 Plus 2.0 microarrays with the Illumina HumanRef-8 BeadArrays. We used a dilution design, where two different RNA samples are mixed at known proportions, and the same RNA is analyzed in duplicate on each platform (see Figure 1). The advantage of this design is that it is very simple to prepare and the number of differentially expressed genes is large, allowing testing of many of the probes on each array. However, in contrast to spike-in studies, the identities of the genes expected to show differential expression are not deﬁnitely known ahead of time. Comparisons between long and short oligonucleotide arrays have been carried out in the past for other array types (3–7). To our knowledge, this study is the ﬁrst to examine the com- parison between in situ synthesized oligonucleotide arrays with bead-based oligonucleotide arrays. Our results show that these two completely different microarray technologies yield, on the whole, very comparable results. This is especially true Nucleic Acids Research, 2005, Vol. 33, No. 18 5915 once the factors of gene expression level and probe placement on the genome are considered. MATERIALS AND METHODS Samples, hybridization and quality control Following informed consent (approved by Cincinnati Children’s Hospital Medical Center Internal Review Board), 50 ml whole blood was collected from 30 adult, apparently healthy, volunteers using Acid Citrate Dextrose as an anti- coagulant. Samples were processed immediately following blood draw. Cells were isolated by Ficoll gradient centrifuga- tion and total RNA was isolated using Trizol (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol, aliquoted and stored at 80 C until use. Whole placenta was collected and immediately frozen in liquid nitro- gen. RNA was extracted using TRI reagent, puriﬁed using RNeasy columns (Qiagen, Valencia, CA), aliquoted and stored at 80 C until use. RNA mixtures (100:0, 95:5, 75:25, 50:50, 25:75, 0:100; PBMC: placenta) were prepared in single ali- quots. Portions of each aliquot were split in half for Affymetrix analysis as technical replicates. Another portion was diluted 1:10 to account for differences in RNA concentration used by each platform and then split into two samples for Illumina analysis as technical replicates. These four ﬁnal samples were sent to the respective facilities for further processing as described below. Quality was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) at their respective core facilities according to internal quality control measures. For Affymetrix microarray analysis, samples were run in the CCHMC Affymetrix core facility. Biotinylated cRNA was synthesized from total RNA (Enzo, Farmingdale, NY). Fol- lowing processing according to the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA), labeled cRNA was quantitated by hybridization to Affymetrix U133plus2.0 GeneChips. For Illumina microarray analysis, samples were prepared and analyzed in Illumina laboratories by Illumina personnel. Biotinylated cRNA was prepared using the Illumina RNA Ampliﬁcation Kit (Ambion, Inc., Austin, TX) according to the manufacturer’s directions starting with 100 ng total RNA. Samples were puriﬁed using the RNeasy kit (Qiagen, Valencia, CA). Hybridization to the Sentrix HumanRef-8 Expression BeadChip (Illumina, Inc., San Diego, CA), wash- ing and scanning were performed according to the Illumina BeadStation 500· manual (revision C). Two BeadChips were used, each one containing eight arrays, so that each dilution series of six samples was run on an individual BeadChip. Data extraction and normalization Affymetrix data were extracted, normalized and summarized with the RMA method from Bioconductor’s ‘affy’ package (8,9), using the default settings. The Illumina data were extra- cted using software provided by the manufacturer. Pilot stud- ies indicated that background subtraction had a negative impact on the Illumina data quality, so we used data that had not been background subtracted. The Illumina data were then normalized using the ‘normalize.quantiles’ function