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Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons.

De Preter K, Pattyn F, Berx G, Strumane K, Menten B, Van Roy F, De Paepe A, Speleman F, Vandesompele J - BMC Genomics (2004)

Bottom Line: Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification.Using this approach, amplification of all previously reported amplified genes in this cell line was detected.Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Medical Genetics, Ghent University Hospital 1K5, De Pintelaan 185, 9000 Gent, Belgium. katleen.depreter@ugent.be

ABSTRACT

Background: Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes.

Results: As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences.

Conclusions: The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.

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Relative expression levels obtained by real-time quantitative RT-PCR: Relative mRNA expression levels obtained by quantitative PCR in 30 neuroblastoma cell lines and 9 normal human tissue samples (samples with gene amplification are marked in red) (relative scale, rescaled to an average expression level of 1).
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Figure 4: Relative expression levels obtained by real-time quantitative RT-PCR: Relative mRNA expression levels obtained by quantitative PCR in 30 neuroblastoma cell lines and 9 normal human tissue samples (samples with gene amplification are marked in red) (relative scale, rescaled to an average expression level of 1).

Mentions: Real-time quantitative PCR was performed in order to analyse the mRNA expression level and gene copy number of novel amplified genes TEM8, g10d12, g10e3, and g4d5, and already known amplified genes MYCN, DDX1, NAG and MEIS1 in 30 NB cell lines and 9 normal human tissue samples (Table 3 and Figure 4). These analyses showed that g10e3 and g4d5 were only amplified and overexpressed in cell line IMR-32. Clone g10d12 was also found to be amplified and overexpressed in cell line SJNB-6. Subsequent gene copy number determination of g10d12 in primary tumour samples indicated a co-amplification frequency with MYCN of 12 % (9/75 tested MYCN amplified tumour samples). The mRNA expression and gene amplification pattern for TEM8 resembles that of MEIS1 ([13] and this study): high expression in a number of cell lines, independent of DNA amplification.


Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons.

De Preter K, Pattyn F, Berx G, Strumane K, Menten B, Van Roy F, De Paepe A, Speleman F, Vandesompele J - BMC Genomics (2004)

Relative expression levels obtained by real-time quantitative RT-PCR: Relative mRNA expression levels obtained by quantitative PCR in 30 neuroblastoma cell lines and 9 normal human tissue samples (samples with gene amplification are marked in red) (relative scale, rescaled to an average expression level of 1).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC365025&req=5

Figure 4: Relative expression levels obtained by real-time quantitative RT-PCR: Relative mRNA expression levels obtained by quantitative PCR in 30 neuroblastoma cell lines and 9 normal human tissue samples (samples with gene amplification are marked in red) (relative scale, rescaled to an average expression level of 1).
Mentions: Real-time quantitative PCR was performed in order to analyse the mRNA expression level and gene copy number of novel amplified genes TEM8, g10d12, g10e3, and g4d5, and already known amplified genes MYCN, DDX1, NAG and MEIS1 in 30 NB cell lines and 9 normal human tissue samples (Table 3 and Figure 4). These analyses showed that g10e3 and g4d5 were only amplified and overexpressed in cell line IMR-32. Clone g10d12 was also found to be amplified and overexpressed in cell line SJNB-6. Subsequent gene copy number determination of g10d12 in primary tumour samples indicated a co-amplification frequency with MYCN of 12 % (9/75 tested MYCN amplified tumour samples). The mRNA expression and gene amplification pattern for TEM8 resembles that of MEIS1 ([13] and this study): high expression in a number of cell lines, independent of DNA amplification.

Bottom Line: Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification.Using this approach, amplification of all previously reported amplified genes in this cell line was detected.Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Medical Genetics, Ghent University Hospital 1K5, De Pintelaan 185, 9000 Gent, Belgium. katleen.depreter@ugent.be

ABSTRACT

Background: Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes.

Results: As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences.

Conclusions: The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.

Show MeSH
Related in: MedlinePlus