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RNA pp 205-217 | Cite as

Validation of RNAi by Real Time PCR

  • Knud Josefsen
  • Ying C. Lee
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 703)

Abstract

Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to verify luciferase mRNA silencing.

Key words

RNA isolation reverse transcription real time PCR LightCycler luciferase shRNA RNAi 

References

  1. 1.
    Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., Conklin, D. S. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16, 948–958.CrossRefPubMedGoogle Scholar
  2. 2.
    Otsuka, Y., Ito, M., Yamaguchi, M., et al. (2002) Enhancement of lipopolysaccharide-stimulated cyclooxygenase-2 mRNA expression and prostaglandin E2 production in gingival fibroblasts from individuals with Down syndrome. Mech Ageing Dev 123, 663–674.CrossRefPubMedGoogle Scholar
  3. 3.
    Fleige, S., Pfaffl, M. W. (2006) RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med 27, 126–139.CrossRefPubMedGoogle Scholar
  4. 4.
    Gilsbach, R., Kouta, M., Bonisch, H., Bruss, M. (2006) Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data. BioTechniques 40, 173–177.CrossRefPubMedGoogle Scholar
  5. 5.
    Nolan, T., Hands, R. E., Bustin, S. A. (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1, 1559–1582.CrossRefPubMedGoogle Scholar
  6. 6.
    Pfaffl, M. W., Georgieva, T. M., Georgiev, I. P., Ontsouka, E., Hageleit, M., Blum, J. W. (2002) Real-time RT-PCR quantification of insulin-like growth factor (IGF)-1, IGF-1 receptor, IGF-2, IGF-2 receptor, insulin receptor, growth hormone receptor, IGF-binding proteins 1, 2 and 3 in the bovine species. Domest Anim Endocrinol 22, 91–102.CrossRefPubMedGoogle Scholar
  7. 7.
    McManus, M. T., Sharp, P. A. (2002) Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3, 737–747.CrossRefPubMedGoogle Scholar
  8. 8.
    Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.CrossRefPubMedGoogle Scholar
  9. 9.
    Henriksen, J. R., Lokke, C., Hammero, M., et al. (2007) Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines. Nucleic Acids Res 35, e67.CrossRefPubMedGoogle Scholar
  10. 10.

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.The Bartholin Institute, Copenhagen University HospitalCopenhagenDenmark
  2. 2.Cellular and Metabolic Research SectionBiomedical Institute, University of CopenhagenCopenhagenDenmark

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