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

Jonson, Tord LU ; Mahlamaki, E H; Karhu, R; Gorunova, Ludmila LU ; Johansson, Bertil LU and Höglund, Mattias LU (2000) In Genes, Chromosomes and Cancer 29(2). p.192-199
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Genes, Chromosomes and Cancer
volume
29
issue
2
pages
192 - 199
publisher
John Wiley & Sons
external identifiers
  • pmid:10959100
  • scopus:0033848780
ISSN
1045-2257
DOI
10.1002/1098-2264(2000)9999:9999<::AID-GCC1026>3.0.CO;2-J
language
English
LU publication?
yes
id
de6c1852-f43c-447e-aa15-0fb327e87525 (old id 1116717)
date added to LUP
2008-07-01 15:48:01
date last changed
2017-01-01 05:10:30
@article{de6c1852-f43c-447e-aa15-0fb327e87525,
  abstract     = {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.},
  author       = {Jonson, Tord and Mahlamaki, E H and Karhu, R and Gorunova, Ludmila and Johansson, Bertil and Höglund, Mattias},
  issn         = {1045-2257},
  language     = {eng},
  number       = {2},
  pages        = {192--199},
  publisher    = {John Wiley & Sons},
  series       = {Genes, Chromosomes and Cancer},
  title        = {Characterization of genomically amplified segments using PCR: optimizing relative-PCR for reliable and simple gene expression and gene copy analyses},
  url          = {http://dx.doi.org/10.1002/1098-2264(2000)9999:9999<::AID-GCC1026>3.0.CO;2-J},
  volume       = {29},
  year         = {2000},
}