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
Part of the Methods in Molecular Biology book series (MIMB, volume 1974)


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.


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


  1. 1.
    Bobbin ML, Rossi JJ (2016) RNA interference (RNAi)-based therapeutics: delivering on the promise? Annu Rev Pharmacol Toxicol 56(1):103–122. Scholar
  2. 2.
    Scherman D, Rousseau A, Bigey P et al (2017) Genetic pharmacology: progresses in siRNA delivery and therapeutic applications. Gene Ther 24(3):151–156. Scholar
  3. 3.
    Yoda M, Kawamata T, Paroo Z et al (2010) ATP-dependent human RISC assembly pathways. Nat Struct Mol Biol 17(1):17–23CrossRefPubMedGoogle Scholar
  4. 4.
    Liu JD, Carmell MA, Rivas FV et al (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305(5689):1437–1441CrossRefPubMedGoogle Scholar
  5. 5.
    Rivas FV, Tolia NH, Song JJ et al (2005) Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol 12(4):340–349CrossRefPubMedGoogle Scholar
  6. 6.
    Martinez J, Patkaniowska A, Urlaub H et al (2002) Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110(5):563–574CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Elbashir SM, Harborth J, Weber K et al (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26(2):199–213CrossRefPubMedGoogle Scholar
  8. 8.
    Nakanishi K (2016) Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins? Wiley Interdiscip Rev RNA 7(5):637–660. Scholar
  9. 9.
    Angart PA, Carlson RJ, Adu-Berchie K et al (2016) Terminal duplex stability and nucleotide identity differentially control siRNA loading and activity in RNA interference. Nucleic Acid Ther 26(5):309–317. Scholar
  10. 10.
    Schwarz D, Hutvagner G, Du T et al (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115(2):199–208CrossRefGoogle Scholar
  11. 11.
    Noland CL, Ma E, Doudna JA (2011) siRNA repositioning for guide strand selection by human dicer complexes. Mol Cell 43(1):110–121CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Sakurai K, Amarzguioui M, Kim D et al (2011) A role for human Dicer in pre-RISC loading of siRNAs. Nucleic Acids Res 39(4):1510–1525CrossRefPubMedGoogle Scholar
  13. 13.
    Ozcan G, Ozpolat B, Coleman RL et al (2015) Preclinical and clinical development of siRNA-based therapeutics. Adv Drug Deliv Rev 87:108–119. Scholar
  14. 14.
    Wittrup A, Lieberman J (2015) Knocking down disease: a progress report on siRNA therapeutics. Nat Rev Genet 16(9):543–552. Scholar
  15. 15.
    Beitzinger M, Meister G (2011) Experimental identification of microRNA targets by immunoprecipitation of Argonaute protein complexes. In: Dalmay T (ed) MicroRNAs in development, vol 732. Humana, Totowa, NJ, pp 153–167CrossRefGoogle Scholar
  16. 16.
    Chen CF, Ridzon DA, Broomer AJ et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Varkonyi-Gasic E, Wu R, Wood M et al (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Tang F, Hajkova P, Barton SC et al (2006) MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res 34(2):e9. Scholar
  19. 19.
    Kramer MF (2011) Stem-loop RT-qPCR for miRNAs. Curr Protoc Mol Biol. Chapter 15:Unit 15.10Google Scholar
  20. 20.
    Jung U, Jiang X, Kaufmann SH et al (2013) A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA. RNA 19(12):1864–1873. Scholar
  21. 21.
    Benes V, Castoldi M (2010) Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods 50(4):244–249. Scholar
  22. 22.
    Czimmerer Z, Hulvely J, Simandi Z et al (2013) A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules. PLoS One 8(1):e55168. Scholar
  23. 23.
    Peltier HJ, Latham GJ (2008) Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues. RNA 14(5):844–852. Scholar
  24. 24.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Caffrey DR, Zhao J, Song Z et al (2011) siRNA off-target effects can be reduced at concentrations that match their individual potency. PLoS One 6(7):e21503CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Kim Y-K, Yeo J, Ha M et al (2012) Retraction notice to: cell adhesion-dependent control of microRNA decay. Mol Cell 46(6):896CrossRefGoogle Scholar

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

Personalised recommendations