number_cancer - Quantification of MYCN, DDX1, and NAG Gene...

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Quantification of MYCN , DDX1 , and NAG Gene Copy Number in Neuroblastoma Using a Real-Time Quantitative PCR Assay Katleen De Preter, M.Sc.,Frank Speleman, Ph.S.Valérie Combaret, Ph.D., John Lunec, Ph.D., Geneviève Laureys, M.D., Ph.D., Bert H.J. Eussen, Nadine Francotte, M.D., Julian Board, Andy D.J. Pearson, M.D., Anne De Paepe, M.D., Ph.D., Nadine Van Roy, Ph.D., Jo Vandesompele, M.Sc. Center for Medical Genetics (KDP, FS, ADP, NVR, JV), and Department of Pediatric Hemato-Oncology (GL), Ghent University Hospital, Ghent, Belgium; Molecular Oncology Unit (VC), Centre Léon Bérard, Lyon, France; Cancer Research Unit (JL, JB), Department of Child Health (AP), University of Newcastle, Newcastle upon Tyne, United Kingdom; Department of Clinical Genetics (BE), Erasmus University, Rotterdam, The Netherlands; and Department of Pediatrics (Hemato-Oncology section) (NF), Cliniques Saint-Joseph Espérance, Montegnée, Belgium Amplification of the proto-oncogene MYCN is a strong adverse prognostic factor in neuroblastoma patients in all tumor stages. The status of the MYCN gene has become an important factor in clinical decision making and therapy stratification. Conse- quently, fast and accurate assessment of MYCN gene copy number is of the utmost importance and the use of two independent methods to determine MYCN status is recommended. For these reasons we have developed and evaluated a real-time quantita- tive PCR (Q-PCR) assay as an alternative for time- consuming Southern blot analysis (SB), and as a second independent technique in parallel with flu- orescence in situ hybridization (FISH) analysis. Ad- vantages of Q-PCR are a large dynamic range of quantification, no requirement for post-PCR sam- ple handling and the need for very small amounts of starting material. The accuracy of the assay was illustrated by measurement of MYCN single gene copy changes in DNA samples of two patients with 2p deletion and duplication, respectively. Two dif- ferent detection chemistries i.e. , a sequence specific TaqMan probe and a generic DNA binding dye SYBR Green I were evaluated and shown to yield similar results. Also, two different calculation methods for copy number determination were used i.e. , the ki- netic method and the comparative C T method, and shown to be equivalent. In total, 175 neuroblastoma samples with known MYCN status, as determined by FISH and/or SB, were examined. Q-PCR data were highly concordant with FISH and SB data. In addition to MYCN copy number evaluation, DDX1 and NAG gene copy numbers were determined us- ing a similar Q-PCR strategy. Survival analysis pointed out that DDX1 and/or NAG amplification has no additional adverse effect on prognosis. KEY WORDS:
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number_cancer - Quantification of MYCN, DDX1, and NAG Gene...

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