[Detection of n-myc gene amplification in neuroblastoma using polymerase chain reaction based methods]

Abstract

We have used semiquantitative and real-time quantitative PCRs to detect n-myc gene-amplification in 21 frozen neuroblastoma biopsies and IMR 32 cell line in order to predict biological behaviour of the tumors. Two primer pairs were used in 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 analysis was 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-labelled fluorescent probe pair to detect the product. Calibration curve, which was set up from a serial dilution including samples with 1, 2, 10, 13, 25-fold n-myc oncogene amplification, was employed for quantitative analysis. Semiquantitative method did not show distinct difference between tumor groups with no amplification and less than 10-fold amplification, while quantitative LightCycler analysis was able to detect even 2-fold amplification. In situ PCRs were performed in two cases of differentiated tumor samples which contained n-myc amplification. We used biotinylated ATP labelling and the same primer pair as for the LightCycler analysis.In both cases differentiated cell forms did not show n-myc gene amplification, while considerable amplification was detected in the neuroblasts.