An improved method for semiquantification of gene amplification from archival material.
A differential PCR-based assay is presented that increases the accuracy of quantification of C-erbB-2 gene-copy number in DNA extracted from archival tumors. The C-erbB-2 gene is amplified in a high percentage of human adenocarcinomas arising at numerous sites, including breast, lung, and stomach. A number of studies have correlated C-erbB-2 with poor prognosis. Gene copy number may be relevant in identifying patients with different clinical outcomes. In this study a target gene and a single copy reference gene were coamplified in the same reaction tube. The level of target gene amplification was reflected by the ratio of the two resulting PCR products. Cell lines exhibiting variable copies ranging from 1 to > 8 of the C-erbB-2 gene were used as quality controls. This technique can reliably show a single copy difference between cell lines and can be used to semiquantitatively estimate gene copy number in DNA extracted from archival paraffin-embedded samples.
Underwood
M A
MA
University Department of Surgery, Glasgow Royal Infirmary, Scotland.
Bartlett
J M
JM
Cooke
T G
TG
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000465:Algorithms; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D002460:Cell Line; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis* / Q000302:isolation & purification; D005260:Female; D018734:Genes, erbB-2; D006801:Humans; D007202:Indicators and Reagents; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000096:biosynthesis*; D015203:Reproducibility of Results; D013274:Stomach Neoplasms / Q000235:genetics / Q000473:pathology; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004273:DNA, Neoplasm; D007202:Indicators and Reagents; D018719:Erb-b2 Receptor Tyrosine Kinases