DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH.
Author(s): Shi SR, Cote RJ, Wu L, Liu C, Datar R, Shi Y, Liu D, Lim H, Taylor CR
Publication: J Histochem Cytochem, 2002, Vol. 50, Page 1005-11
PubMed ID: 12133903 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to compare DNA yield among DNA extraction methods differing in temperature, pH, and use of enzymatic digestion for archival FFPE lymph node, colon, and tonsil specimens.
Summary of Findings:
DNA yield was correlated to both the temperature and pH of the Britton and Robinson type buffer used; with a temperature of 120 degrees C and a pH of 6-9 producing optimal yields. However, optimal results obtained with the newly reported extraction method remained approximately 30% lower than those obtained after enzymatic digestion with proteinase K. DNA quality, determined by OD 260/280 nm wavelength ratios, was equivalent among the heat/alkaline- and enzymatic digestion-based extraction methods.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Normal
- Not specified
Platform:
Analyte Technology Platform DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Antigen retrieval pH 2-12
Analyte Extraction and Purification Temperature of heat-induced retrieval 80 degrees C
100 degrees C
120 degrees C
Analyte Extraction and Purification Analyte isolation method Heat and pH mediated DNA extraction
Proteinase K mediated DNA extraction
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Study Purpose
The purpose of this study was to compare semi-quantitative and real-time kinetic thermocycling PCR results among DNA extraction methods differing in temperature, pH, and use of enzymatic digestion for archival FFPE lymph node, colon, and tonsil specimens.
Summary of Findings:
Semi-quantitative PCR of specimens extracted in an alkaline buffer (pH 6-9) at 120 degrees C produced comparable results to case-matched specimens that underwent proteinase K digestion. Real-time kinetic thermocycling PCR, which estimated the yield of amplifiable genomic DNA, produced more favorable results when specimens were extracted at 120 degrees C in a alkaline buffer of a pH of 9-12 compared to other temperatures, solution pH, and enzymatic digestion.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Normal
- Not specified
Platform:
Analyte Technology Platform DNA PCR DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Antigen retrieval pH 2-12
Analyte Extraction and Purification Temperature of heat-induced retrieval 80 degrees C
100 degrees C
120 degrees C
Analyte Extraction and Purification Analyte isolation method Heat-induced DNA retrieval
Enzymatic digestion DNA retrieval