A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity
A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity
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DOI：
10.1186/1471-2199-15-7
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发表时间：
2014-04-03
影响因子：
--
通讯作者：
Anderl, Jan
中科院分区：
文献类型：
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作者：
Voss, Cristina;Schmitt, Brita;Anderl, Jan
背景：许多关于真核转录机制及其调控的研究依赖于体外实验。传统的RNA聚合酶II转录检测是基于新合成的RNA的放射性标记。结果：本实验室建立了一种新的、无放射性的、可靠的、重复性好的真核细胞体外转录方法。转录后，新合成的RNA被直接检测并使用Quantigene分析进行定量。可选地，可以提纯RNA，并且可以进行引物延伸，然后进行PCR检测或qPCR定量。当被应用于评估RNA聚合酶II抑制剂的活性时，这种新的方法可以准确地估计它们的相对效力。结论：我们的新方法提供了一种标准的体外转录实验的非放射性替代，该方法允许对新转录的、未标记的RNA进行灵敏的检测和精确的定量，并且对于定量像α-Amanitin这样的强转录抑制物特别有用。此外，该方法可以方便地用于定量其他真核生物体外系统的反应产率和转录效率，从而为转录研究领域提供了一个补充工具。
Background: Many studies of the eukaryotic transcription mechanism and its regulation rely on in vitro assays. Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purification and gel electrophoresis is laborious and not always quantitative.Results: Herein, we describe a new, non-radioactive, robust and reproducible eukaryotic in vitro transcription assay that has been established in our laboratory. Upon transcription, the newly synthesized RNA is directly detected and quantified using the QuantiGene assay. Alternatively, the RNA can be purified and a primer extension followed by PCR detection or qPCR quantification can be performed. When applied to assess the activity of RNA polymerase II inhibitors, this new method allowed an accurate estimation of their relative potency.Conclusions: Our novel assay provides a non-radioactive alternative to a standard in vitro transcription assay that allows for sensitive detection and precise quantification of the newly transcribed, unlabelled RNA and is particularly useful for quantification of strong transcriptional inhibitors like alpha-amanitin. Moreover, the method can be easily adapted to quantify the reaction yield and the transcription efficiency of other eukaryotic in vitro systems, thus providing a complementary tool for the field of transcriptional research.