DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution.
Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5-12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.
Shi
Shan-Rong
SR
Department of Pathology, University of Southern California Keck School of Medicine HMR 204, 2011 Zonal Avenue, Los Angeles 90033, USA.
Datar
Ram
R
Liu
Cheng
C
Wu
Lin
L
Zhang
Zina
Z
Cote
Richard J
RJ
Taylor
Clive R
CR
D000941:Antigens / Q000737:chemistry* / Q000302:isolation & purification*; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification*; D005404:Fixatives / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry*; D006361:Heating; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D016707:Tissue Fixation
D000941:Antigens; D005404:Fixatives; D005557:Formaldehyde; D004247:DNA