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Log 10 ( MYCC haploid copy number)

Log 10 ( MYCC haploid copy number). Med8A. D341. D458. EDV. Quantity of amplified cell line (%).

maya-saunders
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Log 10 ( MYCC haploid copy number)

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  1. Log10 (MYCC haploid copy number) Med8A D341 D458 EDV Quantity of amplified cell line (%)

  2. Supplementary Figure 1. Assessment of qPCR assay sensitivity and reproducibility. The sensitivity of the qPCR assay was tested using titration assays, in which increasing proportions (0%, 1%, 2%, 5%, 10%, 25%, 50% and 100%) of known MYCC amplified cell lines (D341 Med, MHH-Med8a and D458 Med [16]) were titrated against decreasing proportions (100%, 99%, 98%, 95%, 90%, 75%, 50% and 0%) of MHH-Med1 (MYCC diploid [16]). The MYCC HCN was measured at each titration point and the minimum quantity of amplified cells required for a sample to be classed as MYCC copy number elevated (i.e. ≥EDV) was determined. The proportion (%) of MYCC amplified medulloblastoma cell line (D458 Med, MHH-Med8a or D341 Med) added to each titration standard is shown on the x-axis. The y-axis shows the MYCC HCN measured in each titration standard using qPCR. The EDV was determined using the reference cohort (Supplementary Table 2), and data shown were obtained using a B2M endogenous reference standard. Cell lines with higher MYCC copy numbers (D458 Med (100 MYCC copies) and MHH-Med8a (80 MYCC copies)) required a lower frequency (%) of cells than cell lines with a lower (D341 Med (20 MYCC copies)) MYCC copy number to reach the EDV threshold. Between 1.5% (using D458 Med or MHH-Med8a cells) and 3.5% (D341 Med cells) of MYCC amplified cells in a given sample produced an elevated MYCC HCN detectable by qPCR. The coefficients of variation (CV) for the slope gradient and line of best fit (r2) of the standard curve, based on all replicates for all samples examined, were; TBP (4.10% and 0.16%), B2M (4.23% and 0.19%), RPLP0 (4.22% and 0.16%), MYCC (4.56% and 0.22%), MYCN (5.62% and 0.36%), and MYCL (4.74% and 0.40%). To further assess reproducibility, MHH-Med1 (MYCC, MYCN and MYCL diploid), D384 Med (MYCC amplified [16]) and IMR-32 (MYCN amplified) cell line controls were added to each independent replicate; results showed strong consistency, the CV of MYCC, MYCN and MYCL HCN in each cell line in all replicates ranged from 9.0%-26.1% (mean, 19.0%).

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