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<p>Methods for absolute quantification of <i>N</i><sup>6</sup>-methyladenosine (m<sup>6</sup>A) have emerged as powerful tools in epitranscriptomics. We previously reported GLORI, a chemical-assisted approach to achieve unbiased and precise m<sup>6</sup>A measurement. However, its lengthy reaction time and severe RNA degradation have limited its applicability, particularly for low-input samples. Here, we present two updated GLORI approaches that are ultrafast, mild and enable absolute m<sup>6</sup>A quantification from one to two orders of magnitude less than the RNA starting material: GLORI 2.0 is compatible with RNA from ~10,000 cells and enhances sensitivity for both transcriptome-wide and locus-specific m<sup>6</sup>A detection; GLORI 3.0 further utilizes a reverse transcription-silent carrier RNA to achieve m<sup>6</sup>A quantification from as low as 500–1,000 cells. Using limited RNA from mouse dorsal hippocampus, we reveal a high modification level in synapse-related gene sets. We envision that the updated GLORI methods will greatly expand the applicability of absolute quantification of m<sup>6</sup>A in biology.</p>

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Mild and ultrafast GLORI enables absolute quantification of m6A methylome from low-input samples

  • 期刊论文

  • 发表日期：

    2025年5月5日

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  • Hanxiao Sun,
  • Bo Lu,
  • Zeyu Zhang,
  • Ye Xiao,
  • Zhe Zhou,
  • Lin Xi,
  • Zhichao Li,
  • Zhe Jiang,
  • Jiayi Zhang,
  • Meng Wang,
  • Cong Liu,
  • Yichen Ma,
  • Jinying Peng,
  • Xiu-Jie Wang,
  • Chengqi Yi

摘要

Methods for absolute quantification of N6-methyladenosine (m6A) have emerged as powerful tools in epitranscriptomics. We previously reported GLORI, a chemical-assisted approach to achieve unbiased and precise m6A measurement. However, its lengthy reaction time and severe RNA degradation have limited its applicability, particularly for low-input samples. Here, we present two updated GLORI approaches that are ultrafast, mild and enable absolute m6A quantification from one to two orders of magnitude less than the RNA starting material: GLORI 2.0 is compatible with RNA from ~10,000 cells and enhances sensitivity for both transcriptome-wide and locus-specific m6A detection; GLORI 3.0 further utilizes a reverse transcription-silent carrier RNA to achieve m6A quantification from as low as 500–1,000 cells. Using limited RNA from mouse dorsal hippocampus, we reveal a high modification level in synapse-related gene sets. We envision that the updated GLORI methods will greatly expand the applicability of absolute quantification of m6A in biology.