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 solid tumors. The size of the amplified regions may vary considerably among individual tumors, and more than one gene may be affected within the same amplicon. The main objective in analyzing genomic amplifications has therefore been to map the shortest region involved and to identify genes with increased 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 L1Hs for gene expression and gene copy number analyses, respectively. We used cDNA derived from pancreatic carcinoma cell lines, and genomic DNA extracted from the same cell lines, as templates in the gene expression and in the gene copy number analyses, respectively. To determine the optimal number of PCR cycles, dilution series of the templates were made. Furthermore, competing primers were used to adjust for differences in target sequence levels. We show that by these simple means it is possible to determine optimal conditions for expression analyses. In addition, the procedure was adapted for the analysis of gene copy number changes at the genomic level using L1Hs as the internal standard. This PCR method makes it possible to produce detailed gene copy number profiles of amplified genomic regions.
Jonson
T
T
Department of Clinical Genetics, University Hospital, Lund, Sweden. Tord.Jonson@klingen.lu.se
Mahlamäki
E H
EH
Karhu
R
R
Gorunova
L
L
Johansson
B
B
Höglund
M
M
D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D005784:Gene Amplification / Q000235:genetics; D018628:Gene Dosage; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D010190:Pancreatic Neoplasms / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000096:biosynthesis; D051901:Smad4 Protein; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D015534:Trans-Activators