An optimized RNA extraction method from archival formalin-fixed paraffin-embedded tissue.
Formalin-fixed and paraffin-embedded (FFPE) tissue is one of the most valuable resources available for molecular biological analysis on tissue after diagnostic histopathological examination. Gene expression profiles of FFPE can provide insights into molecular mechanisms of disease but are difficult due to the high level of cross-linking of biomolecules by formalin fixation. Despite advances in molecular technologies, the quality of RNA obtained from FFPE tissue remains variable. We have optimized a reliable RNA extraction method for FFPE tissue. This approach is based on deparaffinization at high temperature coupled with a 3-day lysis at 65 degrees C. The average total RNA yield is 4.5-5.5 ng per 1 mm(3) 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 nucleotides by bioanalyzer analysis. Although modal lengths of RNA fragments were shorter, reverse transcription and polymerase chain reaction was able to amplify amplicons in the range of 300 base pairs. This optimized method improves the utility of FFPE tissue for molecular profiling studies.
Chung
Joon-Yong
JY
Tissue Array Research Program, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Hewitt
Stephen M
SM
D000818:Animals; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D016612:Paraffin Embedding / Q000379:methods*; D012313:RNA / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016707:Tissue Fixation / Q000379:methods*
D005557:Formaldehyde; D012313:RNA