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Detection of slicer activity by immunopurified plant ARGONAUTE1

Publikation: Bidrag til bog/antologi/rapportBidrag til bog/antologiForskningfagfællebedømt

Small RNA-guided endonucleolysis (“slicing”) of target mRNA is the signature biochemical activity underlying many RNA silencing phenomena. The catalytic slicer activity resides in Argonaute (AGO) proteins. Here, we present two protocols to detect microRNA-guided slicer activity of AGO1 immunopurified from Arabidopsis tissues. The first uses radioactive, cap-labeled RNA substrates produced by in vitro transcription of RNA fragments corresponding to endogenous target sites flanked by 100–200 nucleotides of target sequence. The second protocol uses similarly designed but shorter (around 50 nt) fluorescently labeled RNA. Advantages and disadvantages of the two setups are also discussed.

OriginalsprogEngelsk
TitelPlant MicroRNAs : Methods and Protocols
RedaktørerStefan de Folter
Antal sider22
ForlagHumana Press
Publikationsdato2019
Sider295-316
Kapitel22
ISBN (Trykt)978-1-4939-9041-2
ISBN (Elektronisk)978-1-4939-9042-9
DOI
StatusUdgivet - 2019
NavnMethods in Molecular Biology
Vol/bind1932
ISSN1064-3745

ID: 214511805