Therapeutic Oligonucleotides pp 183-197

Part of the Methods in Molecular Biology book series (MIMB, volume 764)

Quantification of siRNAs In Vitro and In Vivo

  • Angie Cheng
  • Alexander V. Vlassov
  • Susan Magdaleno

Abstract

RNA interference (RNAi) is a regulatory mechanism of eukaryotic cells that uses small interfering RNAs (siRNA) to direct homology-dependent control of gene activity. Applications of RNAi include functional genomics, in vivo target validation, and gene-specific medicines. A key to in vivo application of siRNA is the advancement of efficient delivery to organs, tissues, or cell types of interest. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (post-transfection) and in animals (post- injection). We have adopted the Applied Biosystems TaqMan® based stem–loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these needs. In this chapter, application protocols are described, which enable robust quantification of siRNA, including chemically modified molecules, in vitro and in vivo.

Key words

siRNA shRNA miRNA RNAi quantification PCR TaqMan® siRNA assays 

References

  1. 1.
    Crooke, S. T. (2004) Antisense strategies. Curr. Mol. Med. 4, 465–487.PubMedCrossRefGoogle Scholar
  2. 2.
    Goodchild, J. (2000) Hammerhead ribozymes: biochemical and chemical considerations. Curr. Opin. Mol. Ther. 2, 272–281.PubMedGoogle Scholar
  3. 3.
    Dorsett, Y., and Tuschl, T. (2004) siRNAs: applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 3, 318–329.PubMedCrossRefGoogle Scholar
  4. 4.
    Castanotto, D., and Rossi, J. J. (2009) The promises and pitfalls of RNA-interference-based therapeutics. Nature 457, 426–433.PubMedCrossRefGoogle Scholar
  5. 5.
    Dallas, A., and Vlassov, A. (2006) RNAi: a novel antisense technology and its therapeutic potential. Med. Sci. Monitor 4, 67–74.Google Scholar
  6. 6.
    Dunne, J., Drescher, B., Riehle, H., Hadwiger, P., Young, B. D., Krauter, J., and Heidenreich, O. (2003) The apparent uptake of fluorescently fabeled siRNAs by electroporated cells depends on the fluorochrome. Oligonucleotides 13, 375–380.PubMedCrossRefGoogle Scholar
  7. 7.
    Overhoff, M., Wunsche, W., and Sczakiel, G. (2004) Quantitative detection of siRNA and single-stranded oligonucleotides: relationship between uptake and biological activity of siRNA. Nucleic Acids Res. 32, e170.PubMedCrossRefGoogle Scholar
  8. 8.
    Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E. F., and Hellens, R. P. (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3, 12.PubMedCrossRefGoogle Scholar
  9. 9.
    Ro, S., Park, C., Jin, J., Sanders, K. M., and Yan, W. (2006) A PCR-based method for detection and quantification of small RNAs. Biochem. Biophys. Res. Commun. 351, 756–63.PubMedCrossRefGoogle Scholar
  10. 10.
    Jiang, M., Arzumanov, A. A., Gait, M. J., and Milner, J. (2005) A bi-functional siRNA construct induces RNA interference and also primes PCR amplification for its own quantification. Nucleic Acids Res. 33, e151.PubMedCrossRefGoogle Scholar
  11. 11.
    Sharbati-Tehrani, S., Kutz-Lohroff, B., Bergbauer, R., Scholven, J., and Einspanier, R. (2008) miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample. BMC Mol. Biol. 9, 34.PubMedCrossRefGoogle Scholar
  12. 12.
    Stratford, S., Stec, S., Jadhav, V., Seitzer, J., Abrams, M., and Beverly, M. (2008) Examination of real-time polymerase chain reaction methods for the detection and quantification of modified siRNA. Anal. Biochem. 379, 96–104.PubMedCrossRefGoogle Scholar
  13. 13.
    Chen, C., Ridzon, D. A., Broomer, A. J., Zhou, Z., Lee, D. H., Nguyen, J. T., Barbisin, M., Xu, N. L., Mahuvakar, V. R., Andersen, M. R., Lao, K. Q., Livak, K. J., and Guegler, K. J. (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33, e179.PubMedCrossRefGoogle Scholar
  14. 14.
    Whitehead, K. A., Langer, R., and Anderson, D. G. (2009) Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8, 129–138.PubMedCrossRefGoogle Scholar
  15. 15.
    Manoharan, M. (2004) RNA interference and chemically modified small interfering RNAs. Curr. Opin. Chem. Biol. 8, 570–579.PubMedCrossRefGoogle Scholar
  16. 16.
    Hickerson, R. P., Vlassov, A. V., Leake, D., Wang, Q., Contag, C. H., Johnston, B. H., and Kaspar, R. L. (2008) Stability study of unmodified siRNA and relevance to clinical use. Oligonucleotides 18, 345–354.PubMedCrossRefGoogle Scholar
  17. 17.
    Puri, N., Wang, X., Varma, R., Burnett, C., Beauchamp, L., Batten, D. M., Young, M., Sule, V., Latham, K., Sendera, T., Echeverri, C., Sachse, C., and Magdaleno, S. (2008) LNA incorporated siRNAs exhibit lower off-target effects compared to 2'-OMethoxy in cell phenotypic assays and microarray analysis. Nucleic Acids Symp. Ser. (Oxf.) 52, 25–26.CrossRefGoogle Scholar
  18. 18.
    Cheng, A., Varma, R., and Magdaleno, S. (2009) Your siRNA storage questions answered. Ambion Tech. Notes 16(2), 9–10.Google Scholar
  19. 19.
    Lewis, D. L., and Wolff, J. A. (2005) Delivery of siRNA and siRNA expression constructs to adult mammals by hydrodynamic intravascular injection. Methods Enzymol. 392, 336–350.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Angie Cheng
    • 1
  • Alexander V. Vlassov
    • 2
  • Susan Magdaleno
    • 1
  1. 1.Molecular and Cell Biology DivisionLife TechnologiesAustinUSA
  2. 2.Molecular and Cell Biology DivisionLife TechnologiesAustinUSA

Personalised recommendations