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. 2022 Mar 31;10:42. doi: 10.1186/s40478-022-01335-6

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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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