Relative Quantification of siRNA Strand Loading into Ago2 for Design of Highly Active siRNAs

Methods Mol Biol. 2019:1974:41-56. doi: 10.1007/978-1-4939-9220-1_4.

Abstract

In RNA interference (RNAi), silencing is achieved through the interaction of double-stranded small interfering RNAs (siRNAs) with essential RNAi pathway proteins, including Argonaute 2 (Ago2). Based on these interactions, one strand of the siRNA is loaded into Ago2 forming the active RNA-induced silencing complex (RISC). Optimal siRNAs maximize RISC activity against the intended target and minimize off-target silencing. To achieve the desired activity and specificity, selection of the appropriate siRNA strand for loading into Ago2 is essential. Here, we provide a protocol to quantify the relative loading of individual siRNA strands into Ago2, one factor in determining the capacity of a siRNA to achieve silencing activity and target specificity.

Keywords: Ago2; HeLa; Immunoprecipitation; RT-qPCR; Small RNA; Stem-loop; Transfection; siRNA.

MeSH terms

  • Argonaute Proteins / genetics*
  • Carboxypeptidases / genetics
  • HeLa Cells
  • Humans
  • Neoplasms / genetics*
  • Neoplasms / therapy
  • RNA Interference*
  • RNA, Double-Stranded / genetics
  • RNA, Small Interfering / genetics*
  • Ribonuclease III / genetics

Substances

  • AGO2 protein, human
  • Argonaute Proteins
  • RNA, Double-Stranded
  • RNA, Small Interfering
  • Ribonuclease III
  • Carboxypeptidases
  • SCPEP1 protein, human