RNA expression analysis of formalin-fixed paraffin-embedded tumors

• Published in: Laboratory investigation 2007-04, Vol.87 (4), p.383-391
• Main Authors: Penland, Shannon K, Keku, Temitope O, Torrice, Chad, He, Xiaping, Krishnamurthy, Janakiraman, Hoadley, Katherine A, Woosley, John T, Thomas, Nancy E, Perou, Charles M, Sandler, Robert S, Sharpless, Norman E
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; carcinoma of unknown primary; colorectal cancer; Colorectal Neoplasms - metabolism; DNA microarray; Feasibility Studies; Fixatives; Formaldehyde; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling; Humans; Investigative techniques, diagnostic techniques (general aspects); Laboratory Medicine; Medical sciences; Medicine; Medicine & Public Health; Neoplasms - metabolism; Neoplasms, Unknown Primary - metabolism; Oligonucleotide Array Sequence Analysis; Paraffin Embedding; Pathology; research-article; RNA - biosynthesis; Tissue Fixation
• ISSN: 0023-6837; 1530-0307
• DOI: 10.1038/labinvest.3700529

RNA expression analysis is an important tool in cancer research, but a limitation has been the requirement for high-quality RNA, generally derived from frozen samples. Such tumor sets are often small and lack clinical annotation, whereas formalin-fixed paraffin-embedded (FFPE) materials are abundant. Although RT-PCR-based methods from FFPE samples are finding clinical application, genome-wide microarray analysis has proven difficult. Here, we report expression profiling on RNA from 157 FFPE tumors. RNA was extracted from 2- to 8-year-old FFPE or frozen tumors of known and unknown histologies. Total RNA was analyzed, reverse-transcribed and used for the synthesis of labeled aRNA after two rounds of amplification. Labeled aRNA was hybridized to a 3′-based 22K spot oligonucleotide arrays, and compared to a labeled reference by two-color microarray analysis. After normalization, gene expression profiles were compared by unsupervised hierarchical clustering. Using this approach, at least 24% of unselected FFPE samples produced RNA of sufficient quality for microarray analysis. From our initial studies, we determined criteria based on spectrophotometric analyses and a novel TaqMan-based assay to predict which samples were of sufficient quality for microarray analysis before hybridization. These criteria were validated on an independent set of tumors with a 100% success rate (20 of 20). Unsupervised analysis of informative gene expression profiles distinguished tumor type and subtype, and identified tumor tissue of origin in three unclassified carcinomas. Although only a minority of FFPE blocks could be analyzed, we show that informative RNA expression analysis can be derived from selected FFPE samples.