Profiling RNA polymerase II using the fast chromatin immunoprecipitation method.
Profiling RNA polymerase II using the fast chromatin immunoprecipitation method.
复制标题
使用快速染色质免疫沉淀法分析 RNA 聚合酶 II。
DOI：
10.1007/978-1-59745-248-9_15
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Bomsztyk,Karol
中科院分区：
文献类型：
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作者：
Nelson,Joel;Denisenko,Oleg;Bomsztyk,Karol
用于确定基因转录速率的传统方法，核运行，是耗时的，费力的，并且涉及使用高水平的放射性标记的核苷酸。当与mRNA水平的测量相结合时，RNA聚合酶II（Pol II）染色质免疫沉淀（ChIP）是确定基因转录速率的更简单的替代方法。此外，这种方法提供了更多的信息比核run-on. ChIP检测的权力，它给研究人员的能力，不仅在体内检测特定的蛋白质-DNA相互作用，例如与Pol II，但也确定相对密度的因子沿着基因或整个基因组的转录调控。虽然功能强大，但传统的ChIP测定耗时（涉及2天或更长时间），并且涉及劳动密集型步骤。使用Fast ChIP，我们简化了检测，大大减少了所需的时间和劳动力。改进的测定对于涉及许多样品的研究特别有用，包括同时探测多个转录相关因子和/或观察几个时间点上的转录事件。使用Fast ChIP，可以在5小时内处理24个剪切的染色质样本以产生PCR就绪DNA。
The traditional method for determining the transcription rate of a gene, nuclear run-on, is time consuming, laborious, and involves the use of high levels of radio-labeled nucleotides. When combined with measurements of mRNA levels, RNA polymerase II (Pol II) chromatin immunoprecipitation (ChIP) is a simpler alternative to determine the transcription rate of genes. Moreover, this approach provides more information about the transcriptional regulation of a gene than nuclear run-on. The power of the ChIP assay is that it gives a researcher the ability to not only detect a specific protein–DNA interaction in vivo, for instance with Pol II, but also to determine the relative density of factors along genes or the entire genome. Though powerful, the conventional ChIP assay is time consuming (involving 2 days or more) and involves labor intensive steps. With Fast ChIP we simplified the assay to greatly reduce the time and labor involved. The improved assay is especially useful for studies which involve many samples, including the probing of multiple transcriptionally related factors simultaneously and/or looking at transcription events over several time points. Using Fast ChIP, 24 sheared chromatin samples can be processed to yield PCR ready DNA in 5 h.