RT-PCR-Based Gene Expression Profiling for Cancer Biomarker Discovery from Fixed, Paraffin-Embedded Tissues

  • Aaron Scott
  • Ranjana Ambannavar
  • Jennie Jeong
  • Mei-Lan LiuEmail author
  • Maureen T. Cronin
Part of the Methods in Molecular Biology book series (MIMB, volume 724)


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 words

Breast cancer Formalin-fixed paraffin-embedded tissue Quantitative RT-PCR Expression profiling Molecular biomarkers Prognosis Prediction 


  1. 1.
    Rupp, G.M. and Locker, J. (1988) Purification and analysis of RNA from paraffin-embedded tissues. Biotechniques 6, 56–60.PubMedCrossRefGoogle Scholar
  2. 2.
    Finke, J., Fritzen, R., Ternes, P., Lange, W. and Dolken, G. (1993) An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR. Biotechniques 14, 448–453.PubMedGoogle Scholar
  3. 3.
    Mies, C. (1994) Molecular biological analysis of paraffin-embedded tissues. Hum. Pathol. 25, 555–560.PubMedCrossRefGoogle Scholar
  4. 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
  5. 5.
    Stanta, G. and Bonin, S. (1998) RNA quantitative analysis from fixed and paraffin-embedded tissues: membrane hybridization and capillary electrophoresis. Biotechniques 24, 271–276.PubMedGoogle Scholar
  6. 6.
    Sheils, O.M. and Sweeney, E.C. (1999) TSH receptor status of thyroid neoplasms – TaqMan RT-PCR analysis of archival material. J. Pathol. 188, 87–92.PubMedCrossRefGoogle Scholar
  7. 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
  8. 8.
    Specht, K., Richter, T., Muller, U., Walch, A., Werner, M. and Hofler, H. (2001) Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. Am. J. Pathol. 158, 419–429.PubMedCrossRefGoogle Scholar
  9. 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. 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
  11. 11.
    Haque, T., Faury, D., Albrecht, S., Lopez-Aguilar, E., Hauser, P., Garami, M., et al. (2007) Gene expression profiling from formalin-fixed paraffin-embedded tumors of pediatric glioblastoma. Clin. Cancer Res. 13, 6284–6292.PubMedCrossRefGoogle Scholar
  12. 12.
    Linton, K.M., Hey, Y., Saunders, E., Jeziorska, M., Denton, J., Wilson, C.L., et al. (2008) Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours. Br. J. Cancer 98, 1403–1414.PubMedCrossRefGoogle Scholar
  13. 13.
    Oberli, A., Popovici, V., Delorenzi, M., Baltzer, A., Antonov, J., Matthey, S., et al. (2008) Expression profiling with RNA from formalin-fixed, paraffin-embedded material. BMC Med. Genet. 1, 1–15.CrossRefGoogle Scholar
  14. 14.
    Chang, J., Powles, T.J., Allred, D.C., Ashley, S.E., Clark, G.M., Makris, A., et al. (1999) Biologic markers as predictors of clinical outcome from systemic therapy for primary operable breast cancer. J. Clin. Oncol. 17, 3058–3063.PubMedGoogle Scholar
  15. 15.
    Davis, R.E. and Staudt, L.M. (2002) Molecular diagnosis of lymphoid malignancies by gene expression profiling. Curr. Opin. Hematol. 9, 333–338.PubMedCrossRefGoogle Scholar
  16. 16.
    Gianni, L., Zambetti, M., Clark, K., Baker, J., Cronin, M., Wu, J., et al. (2005) Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J. Clin. Oncol. 23, 7265–7277.PubMedCrossRefGoogle Scholar
  17. 17.
    Golub, T.R., Slonim, D.K., Tamayo, P., Huard, C., Gaasenbeek, M., Mesirov, J.P., et al. (1999) Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 286, 531–537.PubMedCrossRefGoogle Scholar
  18. 18.
    Habel, L.A., Shak, S., Jacobs, M.K., Capra, A., Alexander, C., Pho, M., et al. (2006) A ­population-based study of tumor gene expression and risk of breast cancer death among lymph node-negative patients. Breast Cancer Res. 8, R25.PubMedCrossRefGoogle Scholar
  19. 19.
    Paik, S., Shak, S., Tang, G., Kim, C., Baker, J., Cronin, M., et al. (2004) A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N. Engl. J. Med. 351, 2817–2826.PubMedCrossRefGoogle Scholar
  20. 20.
    Paik, S., Tang, G., Shak, S., Kim, C., Baker, J., Kim, W., et al. (2006) Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer. J. Clin. Oncol. 24, 3726–3734.PubMedCrossRefGoogle Scholar
  21. 21.
    Ramaswamy, S., Ross, K.N., Lander, E.S. and Golub, T.R. (2003) A molecular signature of metastasis in primary solid tumors. Nat. Genet. 33, 49–54.PubMedCrossRefGoogle Scholar
  22. 22.
    Rosenwald, A., Wright, G., Wiestner, A., Chan, W.C., Connors, J.M., Campo, E., et al. (2003) The proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma. Cancer Cell 3, 185–197.PubMedCrossRefGoogle Scholar
  23. 23.
    Sorlie, T., Perou, C.M., Tibshirani, R., Aas, T., Geisler, S., Johnsen, H., et al. (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc. Natl Acad. Sci. USA 98, 10869–10874.PubMedCrossRefGoogle Scholar
  24. 24.
    van de Vijver, M.J., He, Y.D., Veer, L.J., Dai, H., Hart, A.A., Voskuil, D.W., et al. (2002) A gene-expression signature as a predictor of survival in breast cancer. N. Engl. J. Med. 347, 1999–2009.PubMedCrossRefGoogle Scholar
  25. 25.
    Lewis, F., Maughan, N.J., Smith, V., Hillan, K. and Quirke, P. (2001) Unlocking the archive – gene expression in paraffin-­embedded tissue. J. Pathol. 195, 66–71.PubMedCrossRefGoogle Scholar
  26. 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
  27. 27.
    Beqaj, S.H., Flesher, R., Walker, G.R. and Smith, S.A. (2007) Use of the real-time PCR assay in conjunction with MagNA Pure for the detection of mycobacterial DNA from fixed specimens. Diagn. Mol. Pathol. 16, 169–173.PubMedCrossRefGoogle Scholar
  28. 28.
    Ribeiro-Silva, A., Zhang, H. and Jeffrey, S.S. (2007) RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies. BMC Mol. Biol. 8, 118.PubMedCrossRefGoogle Scholar
  29. 29.
    Bohmann, K., Hennig, G., Rogel, U., Poremba, C., Mueller, B.M., Fritz, P., et al. (2009) RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods. Clin. Chem. 55, 1719–1727.PubMedCrossRefGoogle Scholar
  30. 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

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Aaron Scott
    • 1
  • Ranjana Ambannavar
    • 1
  • Jennie Jeong
    • 1
  • Mei-Lan Liu
    • 1
    Email author
  • Maureen T. Cronin
    • 1
  1. 1.Genomic Health, Inc.Redwood CityUSA

Personalised recommendations