RT-PCR-Based Gene Expression Profiling for Cancer Biomarker Discovery from Fixed, Paraffin-Embedded Tissues
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A molecular test providing clear identification of individuals at highest risk for developing metastatic disease from among early stage breast cancer patients has proven to be of great benefit in breast cancer treatment planning and therapeutic management. Patients with high risk of disease recurrence can also get an estimate of the magnitude of benefit to be gained by adding chemotherapy to surgery and hormonal therapy. Developing this clinical test was made possible by the availability of technologies capable of identifying molecular biomarkers from the gene expression profiles of preserved surgical specimens. Molecular tests such as the Oncotype DX® breast cancer test are proving to be more effective tools for individualized patient stratification and treatment planning than traditional methods such as patient demographic variables and histopathology indicators.
Molecular biomarkers must be clinically validated before they can be effectively applied toward patient management in clinical practice. The most effective and efficient means of clinical validation is to use archived surgical specimens annotated with well-characterized clinical outcomes. However, carrying out this type of clinical study requires optimization of traditional molecular expression profiling techniques to analyze RNA from fixed, paraffin-embedded (FPE) tissues. In order to develop our clinically validated breast cancer assay, we modified molecular methods for RNA extraction, RNA quantitation, reverse transcription, and quantitative PCR to work optimally in archived clinical samples. Here, we present an updated description of current best practices for isolating both mRNA and microRNA from FPE tissues for RT-PCR-based expression profiling.
Key wordsBreast cancer Formalin-fixed paraffin-embedded tissue Quantitative RT-PCR Expression profiling Molecular biomarkers Prognosis Prediction
- 4.Krafft, A.E., Duncan, B.W., Bijwaard, K.E., Taubenberger, J.K. and Lichy, J.H. (1997) Optimization of the isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissue: the armed forces institute of pathology experience and literature review. Mol. Diagn. 2, 217–230.PubMedCrossRefGoogle Scholar
- 7.Godfrey, T.E., Kim, S.H., Chavira, M., Ruff, D.W., Warren, R.S., Gray, J.W. et al. (2000) Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction. J. Mol. Diagn. 2, 84–91.PubMedCrossRefGoogle Scholar
- 9.Abrahamsen, H.N., Steiniche, T., Nexo, E., Hamilton-Dutoit, S.J. and Sorensen, B.S. (2003) Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients. J. Mol. Diagn. 5, 34–41.PubMedCrossRefGoogle Scholar
- 10.Cronin, M., Pho, M., Dutta, D., Stephans, J.C., Shak, S., Kiefer, M.C., et al. (2004) Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am. J. Pathol. 164, 35–42.PubMedCrossRefGoogle Scholar
- 26.Cronin, M., Sangli, C., Liu, M.L., Pho, M., Dutta, D., Nguyen, A., et al. (2007) Analytical validation of the Oncotype DX genomic diagnostic test for recurrence prognosis and therapeutic response prediction in node-negative, estrogen receptor-positive breast cancer. Clin. Chem. 53, 1084–1091.PubMedCrossRefGoogle Scholar
- 30.Rozen, S. and Skaletsky, H. (2000) Primer3 on the WWW for general users and for biologist programmers. Meth. Mol. Biol. 132, 365–386.Google Scholar