determine reference intervals in the clinical laboratory; approved guideline. Wayne, PA: National Committee for Clinical Laboratory Standards, 2000. 6. Kudva YC, Sawka AM, Young WF. Clinical review 164: the laboratory diagnosis of adrenal pheochromocytoma: the Mayo Clinic experience [Review]. J Clin Endocrinol Metab 2003;88:4533–9. 7. Bravo EL, Tagle R. Pheochromocytoma: state-of-the-art and future prospects [Review]. Endocr Rev 2003;24:539 –53. DOI: 10.1373/clinchem.2004.035089 Gene Expression Profiles in Formalin-Fixed, Paraffin- Embedded Tissues Obtained with a Novel Assay for Microarray Analysis, Marina Bibikova, 1 Joanne M. Yeakley, 1 Eugene Chudin, 1 Jing Chen, 1 Eliza Wickham, 1 Jessica Wang- Rodriguez, 2 and Jian-Bing Fan 1* ( 1 Illumina, Inc., San Diego, CA; 2 Veterans Affairs Hospital, University of California- San Diego, San Diego, CA 92161; * address correspon- dence to this author at: Illumina, Inc., 9885 Towne Centre Dr., San Diego, CA 92121-1975; fax 858-202-4680, e-mail jfan@illumina.com) Gene expression profiling using microarrays has revolu- tionized the analysis of biological samples. In clinical applications, microarray data have been used to success- fully distinguish among patients exhibiting similar symp- toms (1, 2). Early demonstrations of this power were in the diagnosis of subtypes of acute leukemia (3) and diffuse large B-cell lymphomas (4), and such analyses are gradually gaining acceptance for diagnostic and prognos- tic applications (5–7 ). Investigators are currently accumu- lating microarray data for a broad assortment of such studies but are limited by the requirement of fresh/frozen tissues for sample preparation and labeling (8). This limitation requires the accumulation of fresh samples throughout the course of the disease, which may involve years of monitoring. However, formalin-fixed, paraffin- embedded (FFPE) tissues are widely available and have the advantage of a known patient outcome and drug response history. RNAs derived from these samples are commonly badly degraded and have not been useful for conventional microarray studies (9, 10). We applied a novel expression assay to simultaneously monitor 502 cancer-related genes in RNAs derived from FFPE sam- ples, using microarrays assembled on fiber optic bundles. Our results suggest that this approach can be used for extending microarray analyses to RNAs derived from archival tissue samples. We have recently developed a gene expression method called the DASL TM assay (cDNA-mediated annealing, selection, extension, and ligation) (11 ). This assay targets gene-specific sequences, using pools of chimeric query oligonucleotides. The oligonucleotides all share common primer landing sites so that once the upstream oligonu- cleotide is extended and ligated to the downstream oligo- nucleotide, an amplifiable product is generated. One PCR primer pair is used to amplify all of the amplifiable templates and generate amplicons of similar size (100 bp). This uniformity minimizes potential bias during amplification of many different targets. Currently, the DASL assay can be multiplexed to monitor hundreds of genes (11 ). To allow the use of universal microarrays, the down- stream query oligonucleotides also contain a unique ad- dress sequence that is associated with each gene. This address sequence allows the amplified product, which is labeled during PCR with a fluorescent primer, to hybrid- ize to a microarray bearing the complementary address sequences. This feature provides ready flexibility: to change the genes being monitored, the address sequences can be reassigned and the query oligonucleotide pool resynthesized, using the same arrays. Finally, the cDNA synthesis step is performed with both oligo(dT) and random priming, which frees the assay from dependence on an intact polyA tail, unlike the usual T7 promoter-oligo(dT) priming method for microarray sample preparation (12 ). The use of random hexamers or nonamers in the cDNA synthesis allows representation of targeted cDNA sequences despite RNA degradation. Less than 50% of target probes in FFPE samples were detected when only oligo(dT) primer was used for cDNA synthe- sis, compared with the use of both oligo(dT) and random primers. To monitor gene expression in FFPE samples, we pre- pared RNA from 5-m human tissue sections mounted on microscope slides, obtained from BioChain Institute ac- cording to an Institutional Review Board-approved pro- tocol. Briefly, the slides were deparaffinized in xylenes, and tissue samples were then scraped off with razor blades and held in xylenes until subsequent processing. RNA was extracted by use of the High Pure RNA Paraffin Kit (Roche Applied Science) and quantified by use of RiboGreen (Molecular Probes). When measured on Bio- analyzer (Agilent) RNA Pico Chips, the size of the RNA fragments ranged from 100 to 500 nucleotides, with a peak maximum at 130 nucleotides. On average, 1–2 g of total RNA was isolated from five 5-m tissue sections. For each sample, 200 ng of total RNA was converted to cDNA and processed in the DASL assay as described previously (11, 13). Oligonucleotides targeting 502 can- cer-related genes were used in these experiments, at a density of three nonoverlapping probes per gene, giving a 1506-plex measurement for each sample. Our previous study showed that three probes per gene lend the assay sufficient sensitivity and reproducibility for quantitative detection of differential expression in FFPE tissues (13 ). Mean signal values were computed for each gene by determining the mean signal for the three representative probes (11 ). Because DASL uses random priming in the cDNA synthesis, the probes can be designed to target any unique regions of the gene without the need to limit the selection of optimal probes to the 3' end of transcripts. We profiled 16 FFPE samples of four tissue types— prostate, colon, breast, and lung—with each tissue repre- sented by one nondiseased and several cancer samples. As shown in Fig. 1, the DASL assay gave highly repro- ducible intensity measurements for FFPE samples pre- served for 1.5–3 years. The correlations (r 2 ) between the 2384 Technical Briefs