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

  • Phillip A. Angart
  • Kwasi Adu-Berchie
  • Rebecca J. Carlson
  • Daniel B. Vocelle
  • Christina Chan
  • S. Patrick WaltonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1974)

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

siRNA Ago2 RT-qPCR Small RNA Transfection Immunoprecipitation Stem-loop HeLa 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Phillip A. Angart
    • 1
    • 2
  • Kwasi Adu-Berchie
    • 1
    • 3
  • Rebecca J. Carlson
    • 1
    • 4
  • Daniel B. Vocelle
    • 1
  • Christina Chan
    • 1
  • S. Patrick Walton
    • 1
    Email author
  1. 1.Department of Chemical Engineering and Materials ScienceMichigan State UniversityEast LansingUSA
  2. 2.Office of Biotechnology ProductsU.S. Food and Drug AdministrationSilver SpringUSA
  3. 3.School of Engineering and Applied SciencesHarvard UniversityCambridgeUSA
  4. 4.Harvard-MIT Program in Health Sciences and TechnologyCambridgeUSA

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