1. Quantitative expression profiling of highly degraded RNA from formalin-fixed, paraffin-embedded breast tumor biopsies by oligonucleotide microarrays
- Author
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Alessandra Vigilante, Margherita Mutarelli, Ornella Paris, Maria Ravo, Olì M. V. Grober, Alessandro Weisz, Roberta Tarallo, E. Nola, Luigi Cicatiello, Daniela Cimino, Lorenzo Ferraro, Michele De Bortoli, Ravo, M., Mutarelli, M., Ferraro, L., Grober, O. M. V., Paris, O., Tarallo, R., Vigilante, A., Cimino, D., DE BORTOLI, M., Nola, Ernesto, Cicatiello, L., and Weisz, A.
- Subjects
Microarray ,breast cancer ,formalin-fixed tissues ,microarrays ,Gene Expression ,Biopsy ,Breast Neoplasms ,Biology ,Pathology and Forensic Medicine ,Cell Line, Tumor ,Formaldehyde ,Gene expression ,medicine ,Humans ,RNA, Neoplasm ,Molecular Biology ,Oligonucleotide Array Sequence Analysis ,Paraffin Embedding ,medicine.diagnostic_test ,Microarray analysis techniques ,Gene Expression Profiling ,Carcinoma, Ductal, Breast ,Reproducibility of Results ,RNA ,Cell Biology ,Molecular biology ,In vitro ,Gene expression profiling ,expression profiling ,Female ,DNA microarray ,microarray - Abstract
Microarray-based gene expression profiling is well suited for parallel quantitative analysis of large numbers of RNAs, but its application to cancer biopsies, formalin-fixed paraffin-embedded (FFPE) archived tissues in particular, is limited by the poor quality of the RNA recovered. This represents a serious drawback, since FFPE tumor tissue banks are available with clinical and prognostic annotations, which could be exploited for molecular profiling studies, provided that reliable analytical technologies are found. We applied and evaluated here a microarray-based cDNA-mediated annealing, selection, extension and ligation (DASL) assay for analysis of 502 mRNAs in highly degraded total RNA extracted from cultured cells or FFPE breast cancer biopsies. The study included quantitative and qualitative comparison of data obtained by analysis of the same RNAs with genome-wide oligonucleotide microarrays vs. DASL arrays and, by DASL, before and after extensive in vitro RNA fragmentation. The DASL-based expression profiling assay applied to RNA extracted from MCF-7 cells before or after 24 hours stimulation with a mitogenic dose of 17β-estradiol consistently allowed to detect hormone-induced gene expression changes also following extensive RNA degradation in vitro. Comparable results where obtained with tumor RNA extracted from FFPE breast cancer biopsies (6 to 19 years old). The method proved itself sensitive, reproducible and accurate, when compared with results obtained by microarray analysis of RNA extracted from snap-frozen tissue of the same tumor.
- Published
- 2008