Competitive Polymerase Chain Reaction for the Quantification of N-&lt;i&gt;myc&lt;/i&gt; Gene Copy Number in Neuroblastoma

Inoue, Akira; Hasan, Ziaul; Hemmi, Hiromichi; Kanda, Naotoshi; Hayashi, Yasuhide; Ishizawa, Tohko; Tsuchida, Yoshiaki; Shimatake, Hiroyuki

doi:10.1159/000217988

Cited by 3 publications

(3 citation statements)

References 18 publications

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“…Thus, in its form, PCR assays remain semiquantitative, like the dot blot. An absolute and accurate quantification could be envisaged at the expense of higher complexity i.e., competitive PCR, as proposed by Inoue et al (1996) and microdissection, but justification of such a costly investment is questionable FIGURE 2 -PCR vs. dot blot analysis of N-myc amplification in 98 informative neuroblastoma samples (.20% malignant cells). The N-myc/GAPDH ratio was determined by PCR in tumors characterized as not amplified (NA) or amplified (A) by dot blot.…”

Section: Discussionmentioning

confidence: 99%

Polymerase chain reaction compared with dot blotting for the determination of N-myc gene amplification in neuroblastoma

et al. 1997

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The magnitude of N-myc amplification (NMA) influences the treatment strategy of localized neuroblastomas. Reliable assays are therefore needed for all types of tumor samples. The aim of this comparative study of 119 tumor samples was to determine whether a polymerase chain reaction (PCR)-based assay could replace the current dot blot assay as a routine and reliable means of determining NMA. The 2 assays exhibited comparable sensitivity and were completely concordant for samples containing at least 20% neuroblastoma cells. In their present state, both assays remain semi-quantitative since an absolute quantification of the N-myc copy number in clinical samples is limited by uncertainty about the amplification level of reference cell lines and by the estimation of the proportion of malignant cells. However, PCR offers several advantages over dot blotting, such as feasibility on minute samples, simplicity, standardization, rapidity and cost effectiveness. Int.

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Section: Discussionmentioning

confidence: 99%

Polymerase chain reaction compared with dot blotting for the determination of N-myc gene amplification in neuroblastoma

et al. 1997

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“…After the blots had been washed at a high stringency, filters were exposed on Kodak XAR-5 film with an intensifier screen. Quantification of N-myc copy numbers was carried out using a bio-imaging analyzer, model BAS-2000 (Fuji, Tokyo, Japan), of which details have been described previously (Inoue et al, 1996).…”

Section: Molecular Analysis For N-myc Statusmentioning

confidence: 99%

“…Filters were hybridized with radiolabeled inserts of both pTNB6 and pTNB2. As a control, pHACTB1, which recognizes the sequence for the ␤-actin transcript (donated by Dr. Kato, Kanagawa, Japan) was used (Inoue et al, 1996).…”

Section: Molecular Analysis For N-myc Statusmentioning

confidence: 99%

Extensive genetic heterogeneity in the neuroblastoma cell line NB(TU)1

et al. 1997

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A neuroblastoma cell line displaying genetically unique features was established from a stage III case of a 20‐month‐old girl. Southern blotting by the probe pTNB6, which contains exon 1 of the N‐myc gene, showed that the primary tumor had in total 4 aberrant bands beside the normal amplified band. The established cell line NB(TU)1 had an aberrant N‐myc band (9.0 kb) in addition to the normal band (2.9 kb). Cytogenetic analysis revealed that NB(TU)1 has a composite karyotype composed of at least 7 related karyotypes, which are pseudo‐diploid and contain complex chromosomal abnormalities, including translocations, deletions and homogeneously staining regions (HSRs). Such extensive abnormalities were considered to be prominent among known neuroblastoma cell lines, and it was suggested that NB(TU)1 had acquired a certain type of genetic instability. Analysis of N‐myc bands in 11 clones of NB(TU)1 showed that the intensity ratio of the normal‐sized band (2.9 kb) and the aberrant one (9.0 kb) markedly varied among clones. Moreover, 3 clones showed an additional band with the size of 3.7 kb, which was detectable neither in the parent NB(TU)1 nor in the primary tumor. Thus, NB(TU)1 was shown to be composed of heterogeneous cell components. To further detect such ongoing chromosomal instability, we examined micronuclei formation. NB(TU)1 yielded a larger number of micronuclei than 5 other neuroblastoma cell lines. We conclude that NB(TU)1 has acquired genetic instability detectable by both Southern blotting and cytogenetic analysis. Int. J. Cancer 72:1070–1077, 1997. © 1997 Wiley‐Liss, Inc.

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Detection of N-Myc Gene Amplification in Neuroblastoma by Comparative, in Situ,and Real-Time Polymerase Chain Reaction

Melegh

Bálint

Tóth

et al. 2003

Pediatric Pathology & Molecular Medicine

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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 co-amplify a 520-bp fragment of the beta-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-bp fragment of the n-myc 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 difference 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.

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Competitive Polymerase Chain Reaction for the Quantification of N-<i>myc</i> Gene Copy Number in Neuroblastoma

Cited by 3 publications

References 18 publications

Polymerase chain reaction compared with dot blotting for the determination of N-myc gene amplification in neuroblastoma

Polymerase chain reaction compared with dot blotting for the determination of N-myc gene amplification in neuroblastoma

Extensive genetic heterogeneity in the neuroblastoma cell line NB(TU)1

Detection of N-Myc Gene Amplification in Neuroblastoma by Comparative, in Situ,and Real-Time Polymerase Chain Reaction

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