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A Radiolabeling‐Free, qPCR‐Based Method for Locus‐Specific Pseudouridine Detection
Author(s) -
Lei Zhixin,
Yi Chengqi
Publication year - 2017
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201708276
Subject(s) - pseudouridine , rna , locus (genetics) , melting curve analysis , chemistry , messenger rna , gene , computational biology , microbiology and biotechnology , real time polymerase chain reaction , biology , biochemistry , transfer rna
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.