Global DNA methylation and hydroxymethylation play an important role in gene expression. They can be connected with several diseases. The modification status could be a biomarker to determine the status of disease. A fast, easy and accurate liquid chromatography – tandem mass spectrometry method has been developed for the precise quantitation of 5-methylcytosine and 5-hydroxymethylcytosine. Formic acid was used for the hydrolysis of the DNA strand resulting in nucleobases. These polar hydrolysis products were separated on a normal phase column using reversed phase eluents in inverse gradient mode. Multiple reaction monitoring was applied to achieve high selectivity and sensitivity for the quantitation. A new relative quantitation model was developed by using guanine, as an internal standard, present in samples. The new method was successfully validated with excellent accuracy and precision values in the range of 0.005–0.5% for 5hmC and 1–15% for 5mC. The main advantages of this quantitation method are that, due to relative quantitation, calibration curves can be used without reacquiring the calibration points and no additional isotope labeled internal standards are required. The method was tested to identify the concentrations of 5mC and 5hmC in various sample types. The lowest level of DNA sample required in the case of 0.005% 5hmC is 0.5 μg.

中文翻译：

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一种以鸟嘌呤为内标测定 DNA 甲基化和羟甲基化的相对定量方法

全局 DNA 甲基化和羟甲基化在基因表达中起重要作用。它们可能与多种疾病有关。修饰状态可以是确定疾病状态的生物标志物。一种快速、简单和准确的液相色谱 - 串联质谱法已被开发用于 5-甲基胞嘧啶和 5-羟甲基胞嘧啶的精确定量。甲酸用于水解 DNA 链，产生核碱基。这些极性水解产物在正相柱上使用反相洗脱液以反梯度模式进行分离。应用多反应监测以实现定量的高选择性和灵敏度。通过使用样品中存在的鸟嘌呤作为内标，开发了一种新的相对定量模型。新方法得到了成功验证，其准确度和精密度值范围为 5hmC 的 0.005–0.5% 和 5mC 的 1–15%。这种定量方法的主要优点是，由于相对定量，无需重新获取校准点即可使用校准曲线，并且不需要额外的同位素标记内标。对该方法进行了测试，以确定各种样品类型中 5mC 和 5hmC 的浓度。在 0.005% 5hmC 的情况下，所需的最低 DNA 样品水平为 0.5 μg。无需重新获取校准点即可使用校准曲线，并且不需要额外的同位素标记内标。对该方法进行了测试，以确定各种样品类型中 5mC 和 5hmC 的浓度。在 0.005% 5hmC 的情况下，所需的最低 DNA 样品水平为 0.5 μg。无需重新获取校准点即可使用校准曲线，并且不需要额外的同位素标记内标。对该方法进行了测试，以确定各种样品类型中 5mC 和 5hmC 的浓度。在 0.005% 5hmC 的情况下所需的最低 DNA 样品水平为 0.5 μg。