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(American Journal of Pathology. 2002;160:449-457.)
© 2002 American Society for Investigative Pathology

Technical Advance

Evaluation of Non-Formalin Tissue Fixation for Molecular Profiling Studies

John W. Gillespie*{dagger}, Carolyn J.M. Best{dagger}, Verena E. Bichsel{ddagger}, Kristina A. Cole{dagger}, Susan F. Greenhut§, Stephen M. Hewitt, Mamoun Ahram{dagger}, Yvonne B. Gathright§, Maria J. Merino, Robert L. Strausberg§, Jonathan I. Epstein||, Stanley R. Hamilton**, Gallya Gannot{dagger}{dagger}, Galina V. Baibakova{dagger}, Valerie S. Calvert{ddagger}, Michael J. Flaig{dagger}, Rodrigo F. Chuaqui{dagger}, Judi C. Herring{ddagger}{ddagger}, John Pfeifer§§, Emmanuel F. Petricoin{ddagger}, W. Marston Linehan¶¶, Paul H. Duray, G. Steven Bova|| and Michael R. Emmert-Buck{dagger}¶¶

From the Science Applications InternationalCorporation,*
National Cancer Institute, Frederick,Maryland; the Pathogenetics Unit,{dagger}
theLaboratory of Pathology, National Cancer Institute, Bethesda, Maryland;the Center for Biologics and Research,{ddagger}
Foodand Drug Administration, Bethesda, Maryland; the Cancer Genome AnatomyProject (CGAP),§
Office of the Director,National Cancer Institute, Bethesda, Maryland; the Laboratory ofPathology,
National Cancer Institute, Bethesda,Maryland; the Department of Pathology,||
Johns HopkinsUniversity, Baltimore, Maryland; the Department ofPathology,**
M.D. Anderson Cancer Center,Houston, Texas; the Faculty ofMedicine,{dagger}{dagger}
Tel Aviv University,Tel Aviv, Israel; the Center for Prostate DiseaseResearch,{ddagger}{ddagger}
Rockville, Maryland;the Center for Information Technology,§§
National Institutes of Health, Bethesda, Maryland; and the UrologicOncology Branch,
National CancerInstitute, Bethesda, Maryland

Correspondence: Address correspondence to M.R. Emmert-Buck, Pathogenetics Unit, Laboratory of Pathology and Urologic Oncology Branch, National Cancer Institute, Rm. 2A33, Bldg. 10, 9000 Rockville Pike, Bethesda, MD 20892. E-mail: mbuck\@helix.nih.gov.

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).




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