Real-time quantitative PCR for the measurement of MYCN amplification in human neuroblastoma with the TaqMan detection system

Claudia Casini Raggi, Maria Letizia Bagnoni, Gian Paolo Tonini, Mario Maggi, Giovanna Vona, Pamela Pinzani, Katia Mazzocco, Bruno De Bernardi, Mario Pazzagli, Claudio Orlando

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Neuroblastoma is the most common extracranial malignant solid tumor in children under 5 years and is characterized by a wide clinical and biological heterogeneity, from spontaneously regressive forms to cancers with a rapid and fatal progression. MYCN oncogene amplification is considered the most important prognostic factor to evaluate survival and therapeutic choices in these patients. Methods: Here we present a new assay for rapid and accurate measurement of MYCN amplification, based on real-time quantitative PCR with the TaqMan(TM) reaction. The degree of MYCN amplification was derived from the ratio of the MYCN oncogene and the single-copy reference gene, β-actin. The absolute abundance of these two genes in tumor sample DNA was obtained by extrapolation on external calibration curves generated with reference DNA. Results: We found a variable degree of MYCN amplification, from 2 to 29, in 26 of 49 (53%) neuroblastomas. These results were well correlated to those obtained with a competitive PCR assay in the same samples (r = 0.987). MYCN amplification was associated mainly with advanced cancer stages, and the analysis of overall survival confirmed that the measurement of MYCN amplification is a predictor of patient outcome in neuroblastoma. Patients without MYCN amplification had a cumulative survival significantly higher than patients with low (

Original languageEnglish
Pages (from-to)1918-1924
Number of pages7
JournalClinical Chemistry
Volume45
Issue number11
Publication statusPublished - 1999

ASJC Scopus subject areas

  • Clinical Biochemistry

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