Detection of N-MYC gene amplification in neuroblastoma by comparative, in situ, and real-time polymerase chain reaction

Zsombor Melegh, Ildikó Bálint, Erika Tóth, Erzsébet Csernák, Zoltán Szentirmay, Kálmán Nagy

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Abstract

We have used semiquantitative comparative and real-time quantitative polymerase chain reactions (PCR) to detect n-myc gene-amplification in 20 frozen neuroblastoma biopsies and IMR 32 cell line to predict biological behavior of the tumors. Two primer pairs were used for the semiquantitative method to coamplify a 520-bp fragment of the β-globin gene - used as a single copy reference standard - and a 258-bp fragment of the n-myc gene. After 30 cycles the PCR products were electrophoresed through an agarose gel and were compared to each other with use of a gel-densitometer. Real-time quantitative analyses were performed in a LightCycler instrument. A single primer pair was used to amplify a 120-bpfragment of the nmyc oncogene and a LC640-labeled fluorescent probe pair to detect the product. Calibration curve, set up from a serial dilution including samples with 1, 2, 10, 13, 25-fold n-myc oncogene amplification, was used for quantitative analysis. The semiquantitative method did not show distinct diference between tumor groups with no amplification and less than 10-fold amplification, whereas quantitative LightCycler analysis was able to detect even 2-fold amplification. Differentiated neuroblastomas seldom show n-myc amplification. In spite of this, we have found two partly differentiated tumor samples that contained n-myc amplification. In these cases in situ PCRs were performed to examine the tumor heterogeneity. We used biotinated ATP labeling and the same primer pair as for the LightCycler analysis. In both cases differentiated cells did not show n-myc gene amplification, whereas considerable amplification was detected in the neuroblasts.

Original languageEnglish
Pages (from-to)213-222
Number of pages10
JournalPediatric Pathology and Molecular Medicine
Volume22
Issue number3
DOIs
Publication statusPublished - May 1 2003

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Keywords

  • Comparative PCR
  • In situ PCR
  • N-myc gene
  • Neuroblastoma
  • Real-time PCR

ASJC Scopus subject areas

  • Pediatrics, Perinatology, and Child Health
  • Pathology and Forensic Medicine

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