title: Quantification of oxidized levels of specific RNA species using an aldehyde reactive probe.
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.
authors.affiliation: Reynolds Oklahoma Center on Aging, Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. mikiei.tanaka@usuhs.mil
authors.forename: Mikiei
authors.initials: M
authors.lastname: Tanaka
authors.forename: Song
authors.initials: S
authors.lastname: Han
authors.forename: Pascal A
authors.initials: PA
authors.lastname: Küpfer
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Leumann
authors.forename: William E
authors.initials: WE
authors.lastname: Sonntag
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008863:Microspheres; D015335:Molecular Probes / Q000737:chemistry*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019809:Streptavidin / Q000737:chemistry; D013696:Temperature
chemical_list: D000447:Aldehydes; D015335:Molecular Probes; D012313:RNA; D019809:Streptavidin