title: RNA expression analysis of formalin-fixed paraffin-embedded tumors.
abstract: 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.
authors.affiliation: Department of Medicine, The University of North Carolina School of Medicine, Chapel Hill, NC 27599-7295, USA
authors.forename: Shannon K
authors.initials: SK
authors.lastname: Penland
authors.forename: Temitope O
authors.initials: TO
authors.lastname: Keku
authors.forename: Chad
authors.initials: C
authors.lastname: Torrice
authors.forename: Xiaping
authors.initials: X
authors.lastname: He
authors.forename: Janakiraman
authors.initials: J
authors.lastname: Krishnamurthy
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Hoadley
authors.forename: John T
authors.initials: JT
authors.lastname: Woosley
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Thomas
authors.forename: Charles M
authors.initials: CM
authors.lastname: Perou
authors.forename: Robert S
authors.initials: RS
authors.lastname: Sandler
authors.forename: Norman E
authors.initials: NE
authors.lastname: Sharpless
mesh_terms: D015179:Colorectal Neoplasms / Q000378:metabolism; D005240:Feasibility Studies; D005404:Fixatives; D005557:Formaldehyde; D020869:Gene Expression Profiling; D006801:Humans; D009369:Neoplasms / Q000378:metabolism*; D009382:Neoplasms, Unknown Primary / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding; D012313:RNA / Q000096:biosynthesis*; D016707:Tissue Fixation
chemical_list: D005404:Fixatives; D005557:Formaldehyde; D012313:RNA