Exact quantification of gene amplification in archival tissue sections by laser-assisted microdissection and real-time PCR

Excerpt

Gene amplification is one of the most important mechanisms leading to deregulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is hampered by a mixture of non-neoplastic bystander cells. In order to overcome this shortcoming and to develop an objective quantification method, we have combined laser-based microdissection of tumor cells with the novel 5'-exonuclease based real-time PCR-assay that enables the highly reproducible exact quantification of minute amounts of nucleic acids. As a model system, amplification of the c-erb-B2/Her-2/neu gene and the adjacent topoisomerase IIα gene were determined in paraffin-embedded breast cancer tissue (n = 23) after immunohistochemical labelling and laser-based microdissection. …