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<p>DNA methylation quantification in age-prediction models relies on only a few techniques with inherent limitations and biases, reducing the accuracy and reproducibility. We developed a new droplet digital PCR assay format for accurate DNA methylation quantification, based on three LNA-containing hydrolysis probes to detect methylated and unmethylated alleles. Using a 12-plex assay we simultaneously quantified methylation at six CpGs (<i>ASPA</i>, <i>C1orf132</i>, <i>CCDC102B</i>, <i>EDARADD</i>, <i>ELOVL2</i>, <i>FHL2</i>) in 351 blood samples (age range: 0–95&#xa0;years), showing strong correlations with age (|<i>r|</i>&gt; 0.83). Several age-prediction models were developed, achieving high performance on testing sets (<i>R</i><sup>2</sup>: 0.933–0.973, MAE: 3.18–5.18, RMSE: 4.71–7.27).</p>

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The 12 o’clock assay: an optimized dodecaplex droplet digital PCR assay for robust DNA methylation quantification and epigenetic clock-based age-predictions

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月24日

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  • Ilef Hchaichi,
  • Imène Garali,
  • Alina-Madalina Popa,
  • Antoine Daunay,
  • Nicolas P. Tessier,
  • Mourad Sahbatou,
  • Lise M. Hardy,
  • Aurore Rampanou,
  • Hélène Blanché,
  • Jean-François Zagury,
  • Mathilde Touvier,
  • Hélène Le Buanec,
  • Shufang Renault,
  • Nicolas Girerd,
  • Jean-François Deleuze,
  • Alexandre How-Kit

摘要

DNA methylation quantification in age-prediction models relies on only a few techniques with inherent limitations and biases, reducing the accuracy and reproducibility. We developed a new droplet digital PCR assay format for accurate DNA methylation quantification, based on three LNA-containing hydrolysis probes to detect methylated and unmethylated alleles. Using a 12-plex assay we simultaneously quantified methylation at six CpGs (ASPA, C1orf132, CCDC102B, EDARADD, ELOVL2, FHL2) in 351 blood samples (age range: 0–95 years), showing strong correlations with age (|r|> 0.83). Several age-prediction models were developed, achieving high performance on testing sets (R2: 0.933–0.973, MAE: 3.18–5.18, RMSE: 4.71–7.27).