RNAPol-ChIP:: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription -: art. no. e88
RNAPol-ChIP:: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription -: art. no. e88
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DOI：
10.1093/nar/gnh091
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发表时间：
2004-06-01
影响因子：
14.9
通讯作者：
López-Rodas, G
中科院分区：
文献类型：
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作者：
Sandoval, J;Rodríguez, JL;López-Rodas, G
我们描述了一个程序，RNAPol-ChIP，来测量实际的转录率。它包括通过染色质免疫沉淀（ChIP）检测基因编码区内的RNA聚合酶II。为此，通过PCR分析用聚合酶抗体免疫沉淀的DNA，使用在所需基因的编码区内的扩增子，以避免与暂停在启动子处的聚合酶的干扰。为了验证RNAPol-ChIP，我们将我们的结果与在肝再生或急性胰腺炎期间诱导的几个基因中通过经典方法获得的结果进行比较。当研究短半衰期mRNA基因（例如c-fos和egr 1）时，RNAPol-ChIP给出的结果与其他程序相似。然而，在mRNA更稳定的基因（例如血红素结合蛋白，hpx基因）中，RNAPol-ChIP提供实时转录信息，其结果与核运行或运行的方法相当，这些方法需要分离高度纯化的核。此外，RNAPol-ChIP有利地与基于稳态mRNA分析的方法（北方印迹或RT-PCR）相比较。RNAPol-ChIP的其他优点，如结合经典的ChIP分析，研究转录相关的染色质变化的可能性进行了讨论。
We describe a procedure, RNAPol-ChIP, to measure actual transcriptional rate. It consists of the detection, by chromatin immunoprecipitation (ChIP), of RNA polymerase II within the coding region of genes. To do this, the DNA immunoprecipitated with polymerase antibodies is analysed by PCR, using an amplicon well within the coding region of the desired genes to avoid interferences with polymerase paused at the promoter. To validate RNAPol-ChIP, we compare our results to those obtained by classical methods in several genes induced during either liver regeneration or acute pancreatitis. When short half-life mRNA genes are studied (e.g. c-fos and egr1), RNAPol-ChIP gives results similar to those of other procedures. However, in genes whose mRNA is more stable (e.g. the hemopexin, hpx, gene) RNAPol-ChIP informs on real-time transcription with results comparable to those of methods such as nuclear run-on or run-off, which require the isolation of highly purified nuclei. Moreover, RNAPol-ChIP advantageously compares with methods based on the analysis of steady-state mRNA (northern blot or RT-PCR). Additional advantages of RNAPol-ChIP, such as the possibility of combining it with classical ChIP analysis to study transcription-associated changes in chromatin are discussed.