High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation.
High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation.
复制标题
高通量自动化染色质免疫沉淀作为药物筛选和抗体验证的平台。
DOI：
10.1039/c2lc21290k
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发表时间：
2012
期刊：
影响因子：
6.1
通讯作者：
Quake,StephenR
中科院分区：
文献类型：
--
作者：
Wu,AngelaR;Kawahara,TiaraLA;Rapicavoli,NicoleA;vanRiggelen,Jan;Shroff,EmelynH;Xu,Liwen;Felsher,DeanW;Chang,HowardY;Quake,StephenR
染色质免疫沉淀 (ChIP) 是一种探查蛋白质-DNA 相互作用的检测方法，越来越多地用于药物靶点发现和筛选应用。目前，该实验方案的复杂性和该测定所需的实际操作时间限制了其在低通量应用中的使用；此外，抗体质量的变异性对扩大 ChIP 规模以进行大规模筛选造成了额外的障碍。为了应对这些挑战，我们报告了 HTChIP，这是一种基于微流体的自动化平台，可同时对 16 个不同目标进行高通量 ChIP 筛选测量，并具有进一步扩大规模的潜力。使用 HTChIP，从染色质到可分析的 PCR 结果只需一天，而传统方案则需要几天到一周的时间。 HTChIP 还可用于测试多种抗体，并为下游 ChIP 应用选择性能最佳的抗体，从而节省因抗体质量差而导致 ChIP 测定不成功的时间和试剂成本。我们进行了一系列表征分析，以证明 HTChIP 可以快速、准确地评估细胞的表观遗传状态，并且它足够灵敏，可以在精细的时间分辨率下检测细胞因子刺激剂诱导的表观遗传状态的变化。有了这些结果，我们相信 HTChIP 可以大大改进常规 ChIP、抗体筛选和药物筛选效率，并进一步促进 ChIP 作为研究和发现的宝贵工具。
Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein–DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery.
影响因子：
14.9
作者：
Dahl JA;Collas P
通讯作者：
Collas P
影响因子：
2.9
作者：
DEDON, PC;SOULTS, JA;GOROVSKY, MA
通讯作者：
GOROVSKY, MA
影响因子：
6.1
作者：
Wu AR;Hiatt JB;Lu R;Attema JL;Lobo NA;Weissman IL;Clarke MF;Quake SR
通讯作者：
Quake SR