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Factors in Tissue Handling and Processing That Impact RNA Obtained From Formalin-fixed, Paraffin-embedded Tissue

Joon-Yong Chung, Till Braunschweig, Reginald Williams, Natalie Guerrero, Karl M. Hoffmann, Mijung Kwon, Young K. Song, Steven K. Libutti and Stephen M. Hewitt

Tissue Array Research Program, Laboratory of Pathology (J-YC,TB,RW,NG,SMH), Surgery Branch (MK,SKL), Center for Cancer Research, and Pediatric Oncology Branch (YKS), National Cancer Institute, and Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (KMH), National Institutes of Health, Bethesda, Maryland

Correspondence to: Dr. Stephen M. Hewitt, TARP Lab, Advanced Technology Center, MSC 4605, Bethesda, MD 20892-4605. E-mail: genejock{at}helix.nih.gov

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for molecular assays on tissue after diagnostic histopathological examination. RNA from FFPE tissue suffers from strand breakage and cross-linking. Despite excellent extraction methods, RNA quality from FFPE material remains variable. To address the RNA quality factors within FFPE tissues, we studied RNA quality, isolating individual elements of the tissue fixation and processing including length of fixation in formalin and the type of buffer incorporated in the fixative. We examined the impact of the length of the tissue processing cycle as well. The optimal fixation period of 12–24 hr in phosphate-buffered formalin resulted in better-quality RNA. Longer tissue processing times were associated with higher quality RNA. We determined that the middle region of gene suffers less damage by these processes as shown by real-time quantitative RT-PCR. These data provide key information for the development of methods of analysis of gene expression in archival FFPE tissues and contribute to the establishment of objective standards for the processing and handling of tissue in surgical pathology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 56:1033–1042, 2008)

Key Words: formalin fixed • paraffin embedded • gene expression • RNA integrity • real-time RT-PCR • tissue fixation and processing

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