Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR.
Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR.
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DOI：
10.2144/96206st02
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发表时间：
1996-06
期刊：
影响因子：
2.7
通讯作者：
Zeqi Huang;M. Fasco;M. Fasco;Laurence S. Kaminsky
中科院分区：
文献类型：
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作者：
Zeqi Huang;M. Fasco;M. Fasco;Laurence S. Kaminsky
在竞争性RNA-PCR研究中，污染DNA可能会产生不正确的结果，因为它有可能成为第二个竞争者。使用已公布的方法将DNA作为RNA的污染物来消化DNA，然后在95摄氏度下加热失活5分钟，然后进行逆转录和聚合酶链式反应，初步研究表明，mRNA也受到这些处理的影响。本研究旨在从DNA去除和mRNA保存两个方面对DNA酶I处理RNA进行优化。DT黄递酶转录本的竞争性RNA-聚合酶链式反应被用来量化不同处理的效果。不同初始浓度的其他转录本进行了视觉比较，以确保观察到的影响不是特定mRNAs所特有的。在1U脱氧核糖核酸酶I/微克RNA作用下，该酶在75℃下热变性5min，几乎保留了所有的mRNA。在95℃下热变性5分钟，使大约80%的mRNA失活，而在55℃下加热10分钟并不能完全变性DNA酶I。对于所研究的每个转录本，将1微克RNA与1 U DNA酶在37℃孵育30分钟，然后在75℃热变性5分钟，就足以摧毁所有受污染的DNA，同时完全保存各自的mRNAs。强烈建议将这种处理作为RNA-PCR的常规步骤，特别是与人乳房组织样本(以及可能还有其他人类组织)的竞争性RNA-PCR，这些组织通常被少量基因组DNA污染。
In competitive RNA-PCR studies, contaminating DNA can produce incorrect results because of its potential to act as a second competitor. Preliminary studies using published methods for DNase I digestion of DNA as a contaminant of RNA, followed by thermal inactivation of the enzyme at 95 degrees C for 5 min before reverse transcription and PCR, suggested that the mRNA was also affected by these treatments. This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation. Competitive RNA-PCR of DT-diaphorase transcript was used to quantitate the effects of the various treatments. Other transcripts with varying initial concentrations were visually compared to ensure that the effects observed were not unique to specific mRNAs. With 1 U of DNase I/microgram RNA, thermal denaturation of the enzyme at 75 degrees C for 5 min preserved nearly all of the mRNA. Thermal denaturation at 95 degrees C for 5 min inactivated approximately 80% of the mRNA, whereas heating at 55 degrees C for 10 min did not completely denature the DNase I. For RNA-PCR of every transcript investigated, incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs. This treatment is highly recommended as a routine step in RNA-PCR and particularly with competitive RNA-PCR with human breast tissue samples (and presumably other human tissues), which are often contaminated with small amounts of genomic DNA.