Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for application of diagnostic assays on tissue after microscopic examination. Not only is there a substantial archive of tissue available, but FFPE tissue remains the best method of preparation for microscopic examination in a routine clinical environment. Molecular assays, especially reverse transcription and polymerase chain reaction and expression array-based assays, offer significant potential as diagnostic, prognostic, and predictive tools, but require high quality RNA. Herein, we have optimized a reliable RNA extraction method for FFPE tissue. It is based on deparaffinization at high temperature coupled with a 3-day lysis at 65°C. The average total RNA yield is 4.5 to 5.5 pg per 1 μm3 of archival FFPE tissue, and 260/280 ratios are between 1.80 and 1.95. The extracted RNA has a modal fragment length between 100 and 200 nt by the Bioanalyzer analysis. Although modal lengths of RNA fragments were shorter, reverse transcription and polymerase chain reaction was able to amplify amplicons in range of 300 bp. Pretreatment with RNAlater followed by formalin fixation did not result in improving the RNA quality, but did improve RNA yield. Our method improves the utility of FFPE tissue for molecular profiling studies.
*Tissue Array Research Program, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4605
†PharmacoGenomics Research Center, Inje University College of Medicine, Busan 614-735, Republic of Korea
Reprints: Stephen M. Hewitt, MD, PhD, TARP Lab, Advanced Technology Center, MSC 4605, Bethesda, MD 20892-4605 (e-mail: email@example.com).
Current address: Till Braunschweig, MD, Institute of Pathology, University Clinic, Aachen, Germany.