A systems-level mass spectrometry-based technique for accurate and sensitive quantification of the RNA cap epitranscriptome.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
09 2023
09 2023
Historique:
received:
11
08
2021
accepted:
26
04
2023
medline:
8
9
2023
pubmed:
12
8
2023
entrez:
11
8
2023
Statut:
ppublish
Résumé
Chemical modifications of transcripts with a 5' cap occur in all organisms and function in many aspects of RNA metabolism. To facilitate analysis of RNA caps, we developed a systems-level mass spectrometry-based technique, CapQuant, for accurate and sensitive quantification of the cap epitranscriptome. The protocol includes the addition of stable isotope-labeled cap nucleotides (CNs) to RNA, enzymatic hydrolysis of endogenous RNA to release CNs, and off-line enrichment of CNs by ion-pairing high-pressure liquid chromatography, followed by a 17 min chromatography-coupled tandem quadrupole mass spectrometry run for the identification and quantification of individual CNs. The total time required for the protocol can be up to 7 d. In this approach, 26 CNs can be quantified in eukaryotic poly(A)-tailed RNA, bacterial total RNA and viral RNA. This protocol can be modified to analyze other types of RNA and RNA from in vitro sources. CapQuant stands out from other methods in terms of superior specificity, sensitivity and accuracy, and it is not limited to individual caps nor does it require radiolabeling. Thanks to its unique capability of accurately and sensitively quantifying RNA caps on a systems level, CapQuant can reveal both the RNA cap landscape and the transcription start site distribution of capped RNA in a broad range of settings.
Identifiants
pubmed: 37567932
doi: 10.1038/s41596-023-00857-0
pii: 10.1038/s41596-023-00857-0
doi:
Substances chimiques
RNA Caps
0
RNA, Messenger
0
RNA, Viral
0
RNA, Bacterial
0
Types de publication
Journal Article
Review
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2671-2698Subventions
Organisme : U.S. Department of Health & Human Services | NIH | Center for Information Technology (Center for Information Technology, National Institutes of Health)
ID : ES022858
Organisme : U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)
ID : MH121072
Informations de copyright
© 2023. Springer Nature Limited.
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