Chromatin immunoprecipitation (ChIP): revisiting the efficacy of sample preparation, sonication, quantification of sheared DNA, and analysis via PCR.
Chromatin immunoprecipitation (ChIP): revisiting the efficacy of sample preparation, sonication, quantification of sheared DNA, and analysis via PCR.
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DOI：
10.1371/journal.pone.0026015
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Wamhoff BR
中科院分区：
文献类型：
--
作者：
Schoppee Bortz PD;Wamhoff BR
“定量”ChIP是一种常用于研究细胞和组织中蛋白质-DNA相互作用的工具，是一种经常受到技术错误困扰的困难测定。在此，我们提出了将多个协议合并为快速，可靠和简单的方法所需的过程，以及在进行PCR之前准确定量ChIP DNA的方法。我们证明，高强度超声处理至少30分钟是完全细胞破坏和最大的DNA回收所需的，因为ChIP裂解缓冲液不能裂解甲醛固定的细胞。此外，用chelex-100提取ChIP DNA产生的样品太稀，无法通过纳米分光光度法评估剪切效率或定量。然而，通过苯酚-氯仿-异戊醇（PCIA）方法从Mock-ChIP上清液中提取的DNA可用于评估DNA剪切效率，并用作基于荧光的微孔板测定中的标准。这使得能够在进行实时PCR（rtPCR）之前准确定量螯合物提取的ChIP样品中的DNA并标准化为总DNA浓度。因此，可以在两天内在9个工作台小时内完成的快速ChIP测定已经被沿着以快速、准确和可重复的方式来定量ChIP DNA。所得rtPCR数据更准确地描述了对目标蛋白质-DNA相互作用的处理效果。
The “quantitative” ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest.
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影响因子：
14.9
作者：
van Werven FJ;Timmers HT
通讯作者：
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影响因子：
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作者：
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DOI：
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发表时间：
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期刊：
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作者：
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作者：
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