Evaluation of DNA extraction methods and real time PCR optimization on formalin-fixed paraffin-embedded tissues.
Author(s): Dedhia P, Tarale S, Dhongde G, Khadapkar R, Das B
Publication: Asian Pac J Cancer Prev, 2007, Vol. 8, Page 55-9
PubMed ID: 17477772 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 the yield and quality of DNA generated by four extraction methods for FFPE tissue specimens.
Summary of Findings:
DNA yield from FFPE specimens was greatest using the heat treatment DNA extraction protocol, which also produced DNA of suitable quality for subsequent PCR analysis. The poorest yield was obtained with the phenol/chloroform extraction method, while the protocol reported by Wickham et.al. (2000) generated the highest quality DNA.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Qiagen spin column
Phenol/chloroform
Protocol by Wickham et.al. (2000)
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Study Purpose
The purpose of this study was to determine if amplicon size affects successful PCR analysis of DNA derived from FFPE specimens.
Summary of Findings:
PCR analysis of DNA extracted from FFPE specimens by the heat treatment protocol was successful if the generated amplicon was 250 bp or less. Real-time quantitative PCR optimal annealing temperatures and concentrations for EMSY, Cyclin D1, and beta-globin primers were also reported.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA PCR DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) PCR Specific Length of gene fragment 105 bp
245 bp
476 bp
653 bp
Real-time qPCR Specific Targeted nucleic acid EMSY
Cyclin D1
Beta-globin