Gene Expression Profiles in Formalin-Fixed, Paraffin-Embedded Tissues Obtained with a Novel Assay for Microarray Analysis

Abstract

Gene expression profiling using microarrays has revolutionized the analysis of biological samples. In clinical applications, microarray data have been used to successfully distinguish among patients exhibiting similar symptoms (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 prognostic applications (5)(6)(7). Investigators are currently accumulating 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 samples, 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™ 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 oligonucleotide is extended and ligated to the downstream oligonucleotide, 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 …