Research question

Which is the optimal extraction method for isolating and quantifying circulating cell-free DNA (ccfDNA) from patients with endometriosis? Endometriosis is a common benign disease, associated with pain, infertility and reduced quality of life. Endometriosis is also a known risk factor for various cancers. Robust biomarkers for early detection and prediction of prognosis, however, are lacking. CcfDNA is an easy to obtain biomarker associated with prognosis of cancer patients and enables non-invasive analysis of somatic mutations. Recently, elevated levels of ccfDNA were detected in patients with endometriosis.

Design

Two different ccfDNA extraction methods were compared: Maxwell RSC ccfDNA plasma kit (Maxwell) and QiAamp minElute ccfDNA mini kit (QIAamp). The ccfDNA and circulating mitochondrial DNA (mtDNA) quantities from 34 patients diagnosed with endometriosis were analysed. Fluorometric measurement and quantitative reverse transcription polymerase chain reaction (qRT-PCR) of short and long ALU and mtDNA fragments were used to quantiy ccfDNA.

Results

The yield of ccfDNA isolated with the Maxwell method was significantly higher compared with the QIAamp method (P < 0.0001). Integrity of ccfDNA was significantly higher in the QIAamp isolate (P < 0.0001). Recovered mtDNA was not significantly different between both extraction methods used.

Conclusions

The choice of extraction method can significantly influence the ccfDNA output and integrity. Both methods, however, enabled isolation of sufficient ccfDNA for further downstream applications. With this approach, isolation of ccfDNA could enable the non-invasive detection and analysis of somatic mutation within endometriosis tissue.

中文翻译：

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子宫内膜异位症患者循环游离 DNA 分离和定量方法的比较

研究问题

从子宫内膜异位症患者中分离和定量循环游离细胞 DNA (ccfDNA) 的最佳提取方法是什么？子宫内膜异位症是一种常见的良性疾病，与疼痛、不孕症和生活质量下降有关。子宫内膜异位症也是各种癌症的已知危险因素。然而，缺乏用于早期检测和预测预后的可靠生物标志物。CcfDNA 是一种易于获得的与癌症患者预后相关的生物标志物，可以对体细胞突变进行非侵入性分析。最近，在子宫内膜异位症患者中检测到 ccfDNA 水平升高。

设计

比较了两种不同的 ccfDNA 提取方法：Maxwell RSC ccfDNA 血浆试剂盒 (Maxwell) 和 QiAamp minElute ccfDNA 迷你试剂盒 (QIAamp)。分析了 34 名被诊断患有子宫内膜异位症的患者的 ccfDNA 和循环线粒体 DNA (mtDNA) 数量。短和长ALU和 mtDNA 片段的荧光测量和定量逆转录聚合酶链反应 (qRT-PCR)用于定量 ccfDNA。

结果

与 QIAamp 方法相比，Maxwell 方法分离 ccfDNA 的产量显着高于 QIAamp 方法（P < 0.0001）。QIAamp 分离株中 ccfDNA 的完整性显着更高 ( P < 0.0001)。所使用的两种提取方法之间恢复的 mtDNA 没有显着差异。

结论

提取方法的选择会显着影响 ccfDNA 的输出和完整性。然而，这两种方法都能够分离出足够的 ccfDNA 用于进一步的下游应用。通过这种方法，ccfDNA 的分离可以实现对子宫内膜异位症组织内体细胞突变的非侵入性检测和分析。