Figure 3. MYCN copy number detection in a titration series of mixed neuroblastoma cell lines either harboring or lacking MYCN amplification.

Kelly cells (MYCN-amplified) were titrated with SK-N-AS cells (diploid MYCN) to generate the indicated titration series. Genomic DNA was extracted and fragmented by sonication before MYCN copy number was assessed in 2 ng gDNA using ddPCR (green bars, mean ± SD; n ≥ 3). The number of MYCN copies calculated to be present at each titration step are shown by the white bars. Dashed lines indicate the thresholds used for FISH analysis of the neuroblastoma biopsy in the German NB2004 trial protocol: diploid MYCN (2 copies), MYCN gain (≥ 3 copies) and MYCN amplification (> 8 copies). Target gene copy number was analyzed using QuantaSoft analysis software (version 1.7.4, Bio-Rad). This software determined the copy number by calculating the ratio of the target molecule concentration A (copies/µl) to the reference molecule concentration B (copies/µl) times the number of reference species copies N_B in the human genome $\left(copynumber=\frac{A}{B}\times N_B\right)$.