Rapid, nondenaturing RNA purification using weak anion-exchange fast performance liquid chromatography
Rapid, nondenaturing RNA purification using weak anion-exchange fast performance liquid chromatography
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DOI：
10.1261/rna.1862210
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发表时间：
2010-03-01
期刊：
影响因子：
4.5
通讯作者：
Lukavsky, Peter J.
中科院分区：
文献类型：
--
作者：
Easton, Laura E.;Shibata, Yoko;Lukavsky, Peter J.
我们提供了一种简单、快速的大规模纯化RNA寡核苷酸的方法，适用于生化和结构研究。利用线性化的质粒DNA模板，用T7RNA聚合酶在体外转录RNA。在加入EDTA后，粗转录反应直接用DEAE-Sephose弱阴离子交换层析从所需的RNA产物中分离T7RNA聚合酶、未掺入的rNTPs、小的失败转录本和质粒DNA模板。这种新方法既不需要繁琐的苯酚/氯仿提取T7RNA聚合酶，也不需要RNA变性，这对较大的RNA来说是可取的。此外，同位素标记的rNTPs可以很容易地从柱子中回收，低聚RNA聚集体可以从自然折叠的单体RNA产品中分离出来。
We present a simple and fast method for large-scale purification of RNA oligonucleotides suitable for biochemical and structural studies. RNAs are transcribed in vitro with T7 RNA polymerase using linearized plasmid DNA templates. After addition of EDTA, the crude transcription reaction is subjected directly to weak anion-exchange chromatography using DEAE-sepharose to separate the T7 RNA polymerase, unincorporated rNTPs, small abortive transcripts, and the plasmid DNA template from the desired RNA product. The novel method does neither require tedious phenol/chloroform extraction of the T7 RNA polymerase nor denaturation of the RNA, which is desirable especially for larger RNAs. In addition, isotopically labeled rNTPs can be easily recycled from the column flow-through and oligomeric RNA aggregates can be separated from the natively folded monomeric RNA product.