Reynolds Oklahoma Center on Aging, Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Anal Biochem. 2011 Oct 1;417(1):142-8. doi: 10.1016/j.ab.2011.05.038. Epub 2011 May 30.
Emerging evidence has shown that oxidation of RNA, including messenger RNA (mRNA), is elevated in several age-related diseases, although investigation of oxidized levels of individual RNA species has been limited. Recently we reported that an aldehyde reactive probe (ARP) quantitatively reacts with oxidatively modified depurinated/depyrimidinated (abasic) RNA. Here we report a novel method to isolate oxidized RNA using ARP and streptavidin beads. An oligo RNA containing abasic sites that were derivatized with ARP was pulled down by streptavidin beads, whereas a control oligo RNA was not. In vitro oxidized RNA, as well as total cellular RNA, isolated from oxidatively stressed cells was also pulled down, dependent on oxidation level, and concentrated in the pull-down fraction. Quantitative reverse transcription polymerase chain reaction (RT-PCR) using RNA in the pull-down fraction demonstrated that several gene transcripts were uniquely increased in the fraction by oxidative stress. Thus, our method selectively concentrates oxidized RNA by pull-down and enables the assessment of oxidation levels of individual RNA species.
新兴证据表明，几种与年龄相关的疾病中 RNA（包括信使 RNA（mRNA））的氧化水平升高，尽管对个别 RNA 种类的氧化水平的研究有限。最近我们报道称，醛反应探针（ARP）与氧化修饰的脱嘌呤/脱嘧啶（无碱基）RNA 定量反应。在这里，我们报告了一种使用 ARP 和链霉亲和素珠分离氧化 RNA 的新方法。用 ARP 衍生的含有无碱基位点的寡核苷酸 RNA 被链霉亲和素珠拉下，而对照寡核苷酸 RNA 则没有。体外氧化 RNA，以及从氧化应激细胞中分离的总细胞 RNA，也依赖于氧化水平被拉下并集中在拉下的部分。使用拉下部分的 RNA 进行的定量逆转录聚合酶链反应（RT-PCR）表明，几种基因转录物在应激下通过氧化反应在该部分中独特地增加。因此，我们的方法通过拉下选择性地浓缩氧化 RNA，并能够评估个别 RNA 种类的氧化水平。
