Analysis of gene amplification in archival tissue by differential polymerase chain reaction.
Author(s): Neubauer A, Neubauer B, He M, Effert P, Iglehart D, Frye RA, Liu E
Publication: Oncogene, 1992, Vol. 7, Page 1019-25
PubMed ID: 1349162 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this paper was to develop an optimal DNA extraction method for analysis of oncogene copy number by semi-quantitative differential PCR using FFPE tissues.
Summary of Findings:
Employing normal spleen tissue as negative controls and breast cancer cell lines as positive controls, the authors optimized the differential PCR method for semi-quantitative analysis of the HER2 gene. The authors concluded that xylene and ethanol deparaffinization of 5- to 10-micron sections followed by heat denaturation enabled optimal, consistent differential PCR. Optimal block size was determined to be between 3 and 10 mm diameter.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Standard DNA extraction
High SDS concentration
Proteinase K digestion
Modified deparaffinization
Biospecimen Aliquots and Components Aliquot size/volume < 3 mm diameter block
3-10 mm diameter block
> 10 mm diameter block
5-10 um section thickness
25 um section thickness
50 um section thickness
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Study Purpose
The purpose of this study was to validate the use of differential PCR for detection of oncogene amplification in FFPE tissue with results obtained by slot-blot analysis of case-matched frozen tissue.
Summary of Findings:
Oncogene amplification of HER2 and EGFR was successfully validated by differential PCR using FFPE specimens when a series of DNA quality criteria outlined by the authors was met. These criteria, developed using breast cancer cell lines, were based on ratios of differentially sized amplicons generated by a series of multiplex PCR analyses.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA PCR DNA Dot blot or slot blot Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) PCR Specific Technology platform Differential PCR
Slot blot
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen