A novel automatic quantification method for high-content screening analysis of DNA double strand-break response.
A novel automatic quantification method for high-content screening analysis of DNA double strand-break response.
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一种新型的自动定量方法，用于对DNA双链破裂响应进行高素质筛选分析。
DOI：
10.1038/s41598-017-10063-0
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发表时间：
2017-08-29
影响因子：
4.6
通讯作者：
Feng Y
中科院分区：
文献类型：
--
作者：
Feng J;Lin J;Zhang P;Yang S;Sa Y;Feng Y
高含量筛选通常用于DNA损伤反应的研究。双链断裂(DSB)是最有害的DNA损伤类型之一。用于DSB定量的传统方法是γH_2AX焦点计数，该方法需要手动调整和预设参数，通常被认为不精确、耗时、重复性差和不准确。因此，迫切需要一种稳健的自动替代方法。在本文中，我们提出了一种新的双链断裂定量方法，该方法包括自动图像裁剪、自动焦点分割和荧光强度测量。此外，还增加了一个功能，用于标准化基于共定位分析的DSB反应抑制测量。我们用一种众所周知的DSB反应抑制剂来测试该方法。新方法只需要一个预置参数，从而有效地最小化了与操作员相关的变化。与传统方法相比，新方法检测到不同细胞之间的病灶形成百分比差异更大，从而提高了测量精度。用这种新方法成功地定量了抑制剂对双链断裂反应的影响(p = 0.000)。这种方法在可靠性、自动化和简便性方面的优势表明，它在定量荧光成像研究和对DSB反应中涉及的化合物和因素的高含量筛选方面具有潜力。
High-content screening is commonly used in studies of the DNA damage response. The double-strand break (DSB) is one of the most harmful types of DNA damage lesions. The conventional method used to quantify DSBs is γH2AX foci counting, which requires manual adjustment and preset parameters and is usually regarded as imprecise, time-consuming, poorly reproducible, and inaccurate. Therefore, a robust automatic alternative method is highly desired. In this manuscript, we present a new method for quantifying DSBs which involves automatic image cropping, automatic foci-segmentation and fluorescent intensity measurement. Furthermore, an additional function was added for standardizing the measurement of DSB response inhibition based on co-localization analysis. We tested the method with a well-known inhibitor of DSB response. The new method requires only one preset parameter, which effectively minimizes operator-dependent variations. Compared with conventional methods, the new method detected a higher percentage difference of foci formation between different cells, which can improve measurement accuracy. The effects of the inhibitor on DSB response were successfully quantified with the new method (p = 0.000). The advantages of this method in terms of reliability, automation and simplicity show its potential in quantitative fluorescence imaging studies and high-content screening for compounds and factors involved in DSB response.
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