Ma. Underwood et al., AN IMPROVED METHOD FOR SEMIQUANTIFICATION OF GENE AMPLIFICATION FROM ARCHIVAL MATERIAL, PCR methods and applications, 4(3), 1994, pp. 178-184
A differential PCR-based assay is presented that increases the accurac
y 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 p
oor prognosis. Gene copy number may be relevant in identifying patient
s 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 contro
ls. This technique can reliably show a single copy difference between
cell lines and can be used to semiquantitatively estimate gene copy nu
mber in DNA extracted from archival paraffin-embedded samples.