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

1. 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

   • Tissue - Breast

   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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2. 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

   • Tissue - Breast

   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

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