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
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
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.
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.
Fleige, S., Pfaffl, M. W. (2006) RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med 27, 126–139.
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.
Nolan, T., Hands, R. E., Bustin, S. A. (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1, 1559–1582.
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.
McManus, M. T., Sharp, P. A. (2002) Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3, 737–747.
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.
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.
Editorial, July 2009. http://www.Laboratorytalk.com/News/ida/idal09.html
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Josefsen, K., Lee, Y.C. (2011). Validation of RNAi by Real Time PCR. In: Nielsen, H. (eds) RNA. Methods in Molecular Biology, vol 703. Humana Press. https://doi.org/10.1007/978-1-59745-248-9_14
Download citation
DOI: https://doi.org/10.1007/978-1-59745-248-9_14
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-913-0
Online ISBN: 978-1-59745-248-9
eBook Packages: Springer Protocols