Methods for isolation of cell-free plasma DNA strongly affect DNA yield
Methods for isolation of cell-free plasma DNA strongly affect DNA yield
复制标题
DOI：
10.1016/j.cca.2011.07.011
复制
发表时间：
2011-11-20
影响因子：
5
通讯作者：
Holdenrieder, Stefan
中科院分区：
文献类型：
--
作者：
Fleischhacker, Michael;Schmidt, Bernd;Holdenrieder, Stefan
细胞外核酸存在于血浆、血清和其他体液中，近年来它们的分析受到越来越多的关注。由于血浆和血清中游离DNA的数量少且高度碎片化，快速、高效、可靠的分离方法仍然是一个问题，迄今为止尚未就标准化方法达成一致。我们使用商业供应商的旋转柱（来自 Qiagen 的 QIAamp DNA Blood Midi Kit；来自 Macherey-Nagel 的 NucleoSpin Kit；来自 Roche Diagnostics 的 MagNA Pure 分离系统）在柏林和慕尼黑的实验室中平行分离 44 个血浆样本中的 DNA。所有样品中的 DNA 均通过 LightCycler 480 上的实时 PCR 使用三种不同的靶标（GAPDH、β-珠蛋白、ERV）进行定量。不同分离方法和基因的游离 DNA 数量在中位数 1.6 ng/mL 和 28.1 ng/mL 之间变化。 DNA 绝对值的这种相当大的变化主要是由于使用不同的分离方法造成的 (p
Extracellular nucleic acids are present in plasma, serum, and other body fluids and their analysis has gained increasing attention during recent years. Because of the small quantity and highly fragmented nature of cell-free DNA in plasma and serum, a fast, efficient, and reliable isolation method is still a problem and so far there is no agreement on a standardized method. We used spin columns from commercial suppliers (QIAamp DNA Blood Midi Kit from Qiagen; NucleoSpin Kit from Macherey-Nagel; MagNA Pure isolation system from Roche Diagnostics) to isolate DNA from 44 plasma samples in parallel at laboratories in Berlin and Munich. DNA in all samples was quantified by real-time PCR on a LightCycler 480 using three different targets (GAPDH, beta-globin, ERV). The quantities of cell-free DNA for the different isolation methods and genes varied between medians of 1.6 ng/mL and 28.1 ng/mL. This considerable variation of absolute DNA values was mainly caused by the use of different isolation methods (p