A bisulfite-assisted and ligation-based qPCR amplification technology for locus-specific pseudouridine detection at base resolution.
Nucleic Acids Res
; 52(10): e49, 2024 Jun 10.
Article
en En
| MEDLINE
| ID: mdl-38709875
ABSTRACT
Over 150 types of chemical modifications have been identified in RNA to date, with pseudouridine (Ψ) being one of the most prevalent modifications in RNA. Ψ plays vital roles in various biological processes, and precise, base-resolution detection methods are fundamental for deep analysis of its distribution and function. In this study, we introduced a novel base-resolution Ψ detection method named pseU-TRACE. pseU-TRACE relied on the fact that RNA containing Ψ underwent a base deletion after treatment of bisulfite (BS) during reverse transcription, which enabled efficient ligation of two probes complementary to the cDNA sequence on either side of the Ψ site and successful amplification in subsequent real-time quantitative PCR (qPCR), thereby achieving selective and accurate Ψ detection. Our method accurately and sensitively detected several known Ψ sites in 28S, 18S, 5.8S, and even mRNA. Moreover, pseU-TRACE could be employed to measure the Ψ fraction in RNA and explore the Ψ metabolism of different pseudouridine synthases (PUSs), providing valuable insights into the function of Ψ. Overall, pseU-TRACE represents a reliable, time-efficient and sensitive Ψ detection method.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Seudouridina
/
Sulfitos
/
Reacción en Cadena en Tiempo Real de la Polimerasa
Límite:
Humans
Idioma:
En
Año:
2024
Tipo del documento:
Article