Fig. 6

Detection of CDKN2A tumor suppressor gene deletions in gliomas by ddPCR-based copy number analysis. a Results of Pearson´s correlation analysis of the calculated CDKN2A copy number compared to the detected copy number (CN) in mixtures of U87-MG DNA, which has a homozygous deletion of CDKN2A, and a control DNA that retained both CDKN2A copies. The copy numbers of the undiluted DNA were determined by ddPCR (100% U87-MG DNA: mean CN 0.0; control-DNA: mean CN 1.85 ± 0.02). b Results of CDKN2A copy number analyses using a PrimePCR™ ddPCR copy number assay (Bio-Rad Laboratories) in 66 glioma samples in comparison to other methods (NGS or qPCR). homodel, homozygous deletion; hemidel, hemizygous deletion; ddPCR, droplet digital PCR; NGS, gene panel next generation sequencing [20]; qPCR, semiquantitative real-time PCR. Note that ddPCR-based analysis identified three tumors with homozygous CDKN2A deletion that were considered as showing hemizygous deletions with the other methods. c Fluorescent intensity of the droplets after duplex-PCR using a PrimePCR™ ddPCR copy number assay (Bio-Rad Laboratories) for amplification of a 66 bp-fragment of the CDKN2A locus (upper row) together with self-designed primers for amplification of a 86 bp-reference gene locus (lower row) (NCKAP5, see Additional file 1: Table S2). X axis, number of droplets with fluorescence; Y axis, fluorescence intensity detected in the FAM-channel (Channel 1, blue dots) and HEX-channel (Channel 2, green dots); pink line, threshold; grey dots, dots with background fluorescence of non-incorporated probes; Lane 1, the IDH-mutated astrocytoma, CNS WHO grade 4, exhibited a homozygous deletion of the CDKN2A locus (CN < 0.5). Note that the number of blue droplets are markedly lower than the number of green droplets indicating a homozygous CDKN2A deletion. The few remaining blue droplets were caused by CDKN2A non-deleted cells. Lane 2, the glioblastoma, IDH-wildtype, CNS WHO grade 4, retained both copies of CDKN2A and showed nearly the same numbers of droplets in channel 1 and channel 2. Lane 3, no template control