Characterization of genomically amplified segments using PCR: Optimizing relative-PCR for reliable and simple gene expression and gene copy analyses

Gene amplification is one of the mechanisms for oncogene activation in soli d tumors. The size of the amplified regions may vary considerably among ind ividual tumors, and more than one gene may be affected within the same ampl icon. The main objective in analyzing genomic amplifications has therefore been to map the shortest region involved and to identify genes with increas ed expression as a result of the increased gene copy number. To facilitate such an analysis, we have developed simple polymerase chain reaction (PCR) procedures using the internal standards beta-actin (ACTB) and LI Hs for gen e expression and gene copy number analyses, respectively. We used cDNA deri ved from pancreatic carcinoma cell lines, and genomic DNA extracted from th e same cell lines, as templates in the gene expression and in the gene copy number analyses, respectively. To determine the optimal number of PCR cycl es, dilution series of the templates were made. Furthermore, competing prim ers were used to adjust for differences in target sequence levels. We show that by these simple means it is possible to determine optimal conditions f or expression analyses. in addition, the procedure was adapted for the anal ysis of gene copy number changes at the genomic level using LIHs as the int ernal standard. This PCR method makes it possible to produce detailed gene copy number profiles of amplified genomic regions. (C) 2000 Wiley-Liss, Inc .