Skip to main content

siRNA and DNA Transfer to Cultured Cells

  • Protocol
  • First Online:
Macromolecular Drug Delivery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 480))

Summary

Transfection is a powerful non-viral technology used to deliver foreign nucleic acids into eukaryotic cells, and is the method of choice for a variety of applications including studying the functional role of particular genes and the proteins they code for. By over-expressing genes to produce protein of interest and also by knocking down specific genes, researchers are able to accurately define the role of genes and the protein they encode in various cellular processes. Therefore, this powerful technology is a very vital component of the array of scientific research tools. However, the exact mechanism of action of transfection and also the numerous factors that influence the success of DNA or RNA delivery processes are not clearly understood. Hence, this chapter attempts to explain some of the popular cationic lipid/polymer-based transfection reagents for in vitro DNA/small inhibitory RNA (siRNA) delivery, mainly focusing on the protocols and critical factors to keep in mind to ensure successful delivery of nucleic acids into eukaryotic cells using these methods.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1991) Current Protocols in Molecular Biology, (2nd edn.), Vol. 1. John Wiley and Sons, New York.

    Google Scholar 

  2. Zeyda, M., Borth, N., Kunert, R., and Katinger, H. (1999) Optimization of sorting conditions for the selection of stable, high-producing mammalian cell lines. Biotechnol Prog. 15:953–957.

    Article  PubMed  CAS  Google Scholar 

  3. Agarwal, A., Halvorson, L.M., and Legradi, G. (2005) Pituitary adenylate cyclase-activating polypeptide (PACAP) mimics neuroendocrine and behavioral manifestations of stress: Evidence for PKA-mediated expression of the corticotropin-releasing hormone (CRH) gene. Brain Res Mol Brain Res. 29; 138:45–57.

    Article  Google Scholar 

  4. Dai, H., Hogan, C., Gopalakrishnan, B., Torres-Vazquez, J., Nguyen. M., Park, S., Raftery, L.A., Warrior, R., and Arora, K. (2000) The zinc finger protein schnurri acts as a Smad partner in mediating the transcriptional response to decapentaplegic. Dev Biol. 227:373–387.

    Article  PubMed  CAS  Google Scholar 

  5. Recillas-Targa, F. (2006) Multiple strategies for gene transfer, expression, knockdown, and chromatin influence in mammalian cell lines and transgenic animals. Mol Biotechnol. 34:337–354.

    Article  PubMed  CAS  Google Scholar 

  6. Dileo, J., Miller, T.E., Chesnoy, S., and Huang, L. (2003) Gene transfer to subdermal tissues via a new gene gun design. Hum Gene Ther. 14:79–87.

    Article  PubMed  CAS  Google Scholar 

  7. Xie, T., and Tsong, T.Y. (1993) Study of mechanisms of electric field-induced DNA transfection. V. Effects of DNA topology on surface binding, cell uptake, expression and integration into host chromosomes of DNA in the mammalian cell. Biophys J. 65: 1684–1689.

    Article  PubMed  CAS  Google Scholar 

  8. Pari, G.S., and Xu, Y. (2004) Gene transfer into mammalian cells using calcium phosphate and DEAE-dextran. Methods Mol Biol. 245:25–32.

    PubMed  CAS  Google Scholar 

  9. Graham, F.L., and Van der Eb, A.J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456–467.

    Article  PubMed  CAS  Google Scholar 

  10. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd edn.). Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

    Google Scholar 

  11. Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., and Danielsen, M. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA. 84:7413–7417.

    Article  PubMed  CAS  Google Scholar 

  12. Wei, Y., Pirollo, K.F., Yu, B., Rait, A., Xiang, L., Huang, W., Zhou, Q., Ertem, G., and Chang, E.H. (2004) Enhanced transfection efficiency of a systemically delivered tumor-targeting immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine peptide. Nucleic Acids Res. 32:e48.

    Article  Google Scholar 

  13. Hoekstra, D., Rejman, J., Wasungu, L., Shi, W.F., and Zuhorn, I. (2007) Gene delivery by cationic lipids: in and out of an endosome. Biochemical Society Trans. 35:68–71.

    Article  CAS  Google Scholar 

  14. Kukowska-Latallo, J.F., Bielinska, A.U., Johnson, J., Spindler, R., Tomalia, D.A., and Baker, Jr., J.R. (1996) Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers PNAS. 93:4897–4902.

    Article  PubMed  CAS  Google Scholar 

  15. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411:494–4565.

    Article  PubMed  CAS  Google Scholar 

  16. Gregory, R.I., and Shiekhattar, R. (2005) MicroRNA biogenesis and cancer. Cancer Res. 65:3509–3512.

    Article  PubMed  CAS  Google Scholar 

  17. Vermeulen, A., Behlen, L., Reynolds, A., Wolfson, A., Marshall, W.S., Karpilow, J., and Khvorova, A. (2005) The contributions of dsRNA structure to Dicer specificity and efficiency. RNA. 11:674–682.

    Article  PubMed  CAS  Google Scholar 

  18. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S., and Khvorova, A. (2004) Rational siRNA design for RNA interference. Nat Biotechnol. 22, 326–330.

    Article  PubMed  CAS  Google Scholar 

  19. Tuzmen, S., Kiefer, J., and Mousses, S. (2007) Validation of short interfering RNA knockdowns by quantitative real-time PCR. Methods Mol Biol. 353:177–203.

    PubMed  Google Scholar 

  20. Brazas, R.M., and Hagstrom, J.E. (2005) Delivery of small interfering RNA to mammalian cells in culture by using cationic lipid/polymer-based transfection reagents. Methods Enzymol. 392:112–124.

    Article  PubMed  CAS  Google Scholar 

  21. Persengiev, S.P., Zhu, X., and Green, M.R. (2004) Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs. RNA. 10:12–18.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Gopalakrishnan, B., Wolff, J. (2009). siRNA and DNA Transfer to Cultured Cells. In: Belting, M. (eds) Macromolecular Drug Delivery. Methods in Molecular Biology, vol 480. Humana Press. https://doi.org/10.1007/978-1-59745-429-2_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-429-2_3

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-999-4

  • Online ISBN: 978-1-59745-429-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics