Denaturation and electrophoresis of RNA with glyoxal.
Denaturation and electrophoresis of RNA with glyoxal.
复制标题
用乙二醛对 RNA 进行变性和电泳。
DOI：
10.1101/pdb.prot081000
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发表时间：
2015
影响因子：
--
通讯作者：
D. Rio
中科院分区：
文献类型：
--
作者：
D. Rio
该协议用于变性和分离大mRNA分子（0.5-10 kb）琼脂糖凝胶电泳大小的分离。乙二醛（也称为二甲酰或乙二醛），在本方案中负责维持变性的试剂，含有两个羰基，与鸟嘌呤的亚氨基和氨基反应形成环状结构。它还能与腺嘌呤和胞苷的氨基发生反应。当RNA在乙二醛存在下变性时，这种共价加合物阻止正常的碱基配对，并在琼脂糖凝胶中保持RNA的变性状态。这些加合物一旦形成，在pH <7.0的室温下是稳定的；因此，不需要将乙二醛添加到凝胶或凝胶缓冲液中以保持RNA处于变性状态。由于完全变性的RNA根据其分子质量在琼脂糖凝胶中迁移，因此该方法可用于准确地确定mRNA分子的大小。在电泳和乙草醛化逆转后，RNA可以使用北杂交程序检测到。
This protocol is used to denature and separate large mRNA molecules (0.5-10 kb) on agarose gels by electrophoretic size fractionation. Glyoxal (also called diformyl or ethanedial), the agent responsible for maintaining denaturation in this protocol, contains two carbonyl groups that react to form a cyclic ring structure with the imino and amino groups of guanine. It can also react with the amino groups of adenine and cytidine. When RNA is denatured in the presence of glyoxal, this covalent adduct prevents normal base pairing and maintains the RNA in a denatured state in agarose gels. Once formed, these adducts are stable at room temperature at pH <7.0; thus, there is no need to add glyoxal to the gel or to the gel buffers to maintain the RNA in the denatured state. Because the fully denatured RNA migrates through agarose gels according to its molecular mass, this method can be used to accurately size mRNA molecules. Following electrophoresis and reversal of glyoxalation, the RNA can be detected using a northern hybridization procedure.