Summary
RNA interference (RNAi) is a powerful, quick, and easy technique to reduce the expression of a particular gene. However, investigators need to consider several steps for the experiments, including the design of the siRNA, an efficient delivery method, and a means for monitoring the biological effects of the siRNA introduced. Adipocytes have long been recognized as one of the most difficult cell types in which to perform gene delivery. In this chapter, three distinct transfec-tion methods to obtain high efficiency of gene delivery and gene knockdown are described. The transfection efficiency of siRNA and shRNA is also compared.
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 subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431:343–349
Echeverri CJ, Perrimon N (2006) High-throughput RNAi screening in cultured cells: a user's guide. Nat Rev Genet. 7:373–384
Elbashir SM, Harborth J, Weber K, Tuschl T (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26:199–213
Pei Y, Tuschl T (2006) On the art of identifying effective and specific siRNAs. Nat Methods 3:670–676
Sontheimer EJ (2005) Assembly and function of RNA silencing complexes. Nat Rev Mol Cell Biol 6:127–138
Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes Dev 19:517–529
Filipowicz W, Jaskiewicz L, Kolb FA, Pillai RS (2005) Post-transcriptional genesilencing by siRNAs and miRNAs. Curr Opin Struct Biol 15:331–341
Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R (2006) Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev 20:515–524
Reynolds A, Anderson E M., Vermeulen A, Fedorov Y, Robinson K, Leake D, Karpilow J, Marshall WS, Khvorova A (2006) Induction of the interferon response by siRNA is cell type and duplex length dependent. RNA 12:988–993
Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD (1998) How cells respond to interferons. Annu Rev Biochem 67:227–264
Aoyagi T, Shimba S, Tezuka M (2005) Characteristics of circadian gene expressions in mice white adipose tissue and 3T3-L1 adipocytes. J Health Sci 51:21–32
Shimba S, Ishii N, Ohta Y, Ohno T, Watabe Y, Hayashi M, Wada T, Aoyagi T, Tezuka M (2005) BMAL1, a component of the molecular clock, regulates adipogenesis. Proc Natl Acad Sci U S A 102:12071–12076
Acknowledgments
This work is supported in part by a grant from the Ministry of Education, Sciences, Sports, and Culture of Japan; a General Individual Research grant from Nihon University; a grant from Ono Medical Research Foundation; an Interdisciplinary General Joint Research grant from Nihon University; and the Academic Frontier Project for Private Universities, a matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology 2002–2006.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Shimba, S. (2008). Application of RNA Interference Techniques to Adipose Cell Cultures. In: Yang, K. (eds) Adipose Tissue Protocols. Methods in Molecular Biology™, vol 456. Humana Press. https://doi.org/10.1007/978-1-59745-245-8_18
Download citation
DOI: https://doi.org/10.1007/978-1-59745-245-8_18
Publisher Name: Humana Press
Print ISBN: 978-1-58829-916-1
Online ISBN: 978-1-59745-245-8
eBook Packages: Springer Protocols