ArticleMolecular diagnosis & therapy2021
Development and Evaluation of a Droplet Digital PCR Assay for 8p23 β-Defensin Cluster Copy Number Determination.
Article in Molecular diagnosis & therapy, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed, 9 citations in OpenAlex.
- Influence of DEFB4 copy number variants on gene expression in monocytes.BMC research notes · 2025Article
- Digital droplet PCR is an accurate and precise method to measure DNA copy number.Scientific reports · 2025Article
- Highly multiplexed digital PCR assay for simultaneous quantification of variant allele frequencies and copy number alterations of KRAS and GNAS in pancreatic cancer precursors.Molecular oncology · 2025Article
- Droplet digital PCR assay for precise determination of FRS2 gene copy number in bladder cancer.BMC cancer · 2025Article
- Low C4A copy numbers and higher HERV gene insertion contributes to increased risk of SLE, with absence of association with disease phenotype and disease activity.Immunologic research · 2024Article
- Digital droplet PCR versus quantitative PCR for lipoprotein (a) kringle IV type 2 repeat polymorphism genetic characterization.Journal of clinical laboratory analysis · 2024Article
- Digital droplet PCR analysis of organoids generated from mouse mammary tumors demonstrates proof-of-concept capture of tumor heterogeneity.Frontiers in cell and developmental biology · 2024Article
- Reference gene selection for clinical chimeric antigen receptor T-cell product vector copy number assays.Cytotherapy · 2023Article
- New insights into the role of ribonuclease P protein subunit p30 from tumor to internal reference.Frontiers in oncology · 2022Review
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Authors and funding
5 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
introductionIt has been proposed that the copy number (CN) variation (CNV) in β-defensin genes (DEFB) on human chromosome 8p23 determines phenotypic differences in inflammatory diseases. However, no method for accurate and easy DEFB CN quantification is yet available.
objectiveDroplet digital polymerase chain reaction (ddPCR) is a novel method for CNV quantification and has been used for genes such as CCL4L, CCL3L1, AMY1, and HER2. However, to date, no ddPCR assay has been available for DEFB CN determination. In the present study, we aimed to develop and evaluate such a ddPCR assay.
methodsThe assay was designed using DEFB4 and RPP30 as the target and the reference gene, respectively. To evaluate the assay, 283 DNA samples with known CNs previously determined using the multiple ligation-dependent probe amplification (MLPA) method, the current gold standard, were used as standards. To discover the optimal DNA template amount, we tested 80 to 2.5 ng DNA by a serial of one to two dilutions of eight samples. To evaluate the reproducibility of the assay, 31 samples were repeated to calculate the intra- and inter-assay variations. To further validate the reliability of the assay, the CNs of all 283 samples were determined using ddPCR. To compare results with those using quantitative PCR (qPCR), DEFB CNs for 48 samples were determined using qPCR with the same primers and probes.
resultsIn a one-dimensional plot, the positive and negative droplets were clearly separated in both DEFB4 and RPP30 detection channels. In a two-dimensional plot, four populations of droplets were observed. The 20 ng template DNA proved optimal, with either high (80 ng) or low (10, 5, 2.5 ng) template input leading to ambiguous or inaccurate results. For the 31 standard samples, DEFB CNs were accurately determined with small intra- and inter-assay variations (coefficient of variation < 0.04 for both). In the validation cohort, ddPCR provided the correct CN for all 283 samples with high confidence. qPCR measurements for the 48 samples produced noisy data with high uncertainty and low accuracy.
conclusionsddPCR is an accurate, reproducible, easy-to-use, cheap, high-throughput method for DEFB CN determination. ddPCR could be applied to DEFB CN quantification in large-scale case-control studies.
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