Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells

STAR Protoc. 2022 Jul 31;3(3):101596. doi: 10.1016/j.xpro.2022.101596. eCollection 2022 Sep 16.

Abstract

Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lysates for RISC assembly assay. We then detail AGO1 complexes immunoprecipitation for RISC cleavage assay. This protocol can detect RISC assembly and cleavage products within 5 days. Moreover, it can detect 5'- and 3'-cleavage products simultaneously. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022).

Keywords: Cell-based Assays; Gene Expression; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry.

MeSH terms

  • Animals
  • Argonaute Proteins / genetics
  • Drosophila / genetics
  • Drosophila Proteins* / genetics
  • RNA
  • RNA Interference
  • RNA-Induced Silencing Complex* / genetics

Substances

  • AGO1 protein, Drosophila
  • Argonaute Proteins
  • Drosophila Proteins
  • RNA-Induced Silencing Complex
  • RNA