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Quantification of oxidized levels of specific RNA species using an aldehyde reactive probe

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Publication date 2011
Author Tanaka Mikiei, Han Song, Küpfer Pascal A., Leumann Christian J., Sonntag William E.,
Project Chemically modified Oligonucleotides for Biotechnology and Material Sciences
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Original article (peer-reviewed)

Journal Analytical Biochemistry
Volume (Issue) 417
Page(s) 142 - 148
Title of proceedings Analytical Biochemistry

Abstract

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.
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