Comparison of the DNA Extraction Methods for Polymerase Chain Reaction Amplification from Formalin-Fixed and Paraffin-Embedded Tissues.
Author(s): Sato Y, Sugie R, Tsuchiya B, Kameya T, Natori M, Mukai K
Publication: Diagn Mol Pathol, 2001, Vol. 10, Page 265
PubMed ID: 11763318 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
Comparative analysis of DNA yield and size distribution using six different DNA extraction methods for FFPE tissue.
Summary of Findings:
DNA extraction methods using nonorganic solutions yielded a greater quantity of DNA , 1.5-fold greater, than more traditional phenol-chloroform methods. The lowest DNA yield was observed with microwave irradiation, protein digestion, and phenol-chloroform purification. Further, the size distribution of DNA extracted using nonorganic methods was shifted, with a greater abundance of shorter molecules than observed with organic solution extraction methods.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Lymphoma
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Deparaffinization Conducted at room temperature
Conducted at 80 degrees C
Conducted via microwave irradiation
Xylene
Hemo Clear
Nonorganic digestion buffer
Analyte Extraction and Purification Analyte isolation method Phenol-chloroform
Nonorganic digestion buffer
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Study Purpose
To assess the applicability of DNA extracted from FFPE tissue by different methods for PCR analysis of different size amplicons of the beta-globin gene.
Summary of Findings:
PCR amplification of the largest product (989 bp) was unsuccessful, with the exception of the microwave irradiation/ nonorganic DNA extraction method, while amplification levels of shorter products (110 and 268 bp) were comparable among extraction techniques. Amplification of the 536 bp product was extraction method dependent, with the microwave irradiation/ nonorganic DNA extraction method yielding the most product.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Lymphoma
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Deparaffinization Conducted at room temperature
Conducted at 80 degrees C
Conducted via microwave irradiation
Xylene
Hemo Clear
Nonorganic digestion buffer
Analyte Extraction and Purification Analyte isolation method Phenol-chloroform
Nonorganic digestion buffer
PCR Specific Targeted nucleic acid beta-globin
PCR Specific Length of gene fragment 110 bp
268 bp
536 bp
989 bp
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Study Purpose
To determine the applicability of DNA extracted from FFPE tissue using microwave irradiation and nonorganic DNA isolation for the PCR-single strand confirmation polymorphism (SSCP) assay of the beta-catenin gene.
Summary of Findings:
PCR-SSCP analysis of exon 3 of the beta-catenin gene was successful in all six cases examined, with no alterations observed. Results were confirmed by direct sequencing.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Lymphoma
Platform:
Analyte Technology Platform DNA DNA sequencing DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) PCR Specific Targeted nucleic acid beta-catenin