Pseudouridine (Ψ) is the most abundant post‐transcriptional RNA modification. Methods have been developed for locus‐specific Ψ detection; however, they often involve radiolabeling of RNA, require advanced experimental skills, and can be time‐c...
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https://www.riss.kr/link?id=O120443911
2017년
-
0044-8249
1521-3757
학술저널
15074-15078 [※수록면이 p5 이하이면, Review, Columns, Editor's Note, Abstract 등일 경우가 있습니다.]
0
상세조회0
다운로드다국어 초록 (Multilingual Abstract)
Pseudouridine (Ψ) is the most abundant post‐transcriptional RNA modification. Methods have been developed for locus‐specific Ψ detection; however, they often involve radiolabeling of RNA, require advanced experimental skills, and can be time‐c...
Pseudouridine (Ψ) is the most abundant post‐transcriptional RNA modification. Methods have been developed for locus‐specific Ψ detection; however, they often involve radiolabeling of RNA, require advanced experimental skills, and can be time‐consuming. Herein we report a radiolabeling‐free, qPCR‐based method to rapidly detect locus‐specific Ψ. Pseudouridine residues were labeled chemically, and the resulting adducts induced mutation/deletion during reverse transcription (RT) to generate qPCR products with different melting temperatures and hence altered melting curves. We validated our method on known Ψ sites in rRNA and then used it to sensitively detect Ψ residues in lncRNA and mRNA of low abundance. Finally, we applied our method to pseudouridine synthase identification and showed that Ψ616 in PSME2 mRNA is dependent on PUS7. Our facile and cost‐effective method takes only 1.5 days to complete, and with slight adjustment it can be applied to the detection of other epitranscriptomic marks.
Auf Spurensuche: Eine schnelle und kostengünstige Methode für den Nachweis der verbreiteten posttranskriptionalen RNA‐Modifikation Pseudouridin im humanen Transkriptom wurde entwickelt. Pseudouridin wird chemisch markiert, und die resultierenden Addukte induzieren Mutationen/Deletionen bei der reversen Transkription (RT), um qPCR‐Produkte mit unterschiedlichen Schmelztemperaturen und somit veränderten Schmelzkurven zu generieren.
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