MicroRNA Knock Down by Cholesterol-Conjugated Antisense Oligos in Mouse Organ Culture

  • Sharon Kredo-Russo
  • Eran HornsteinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 732)


Here, we detail a protocol to design and introduce sequence-specific cholesterol-conjugated antisense oligonucleotides into mouse organ culture. We review design principles for “antagomirs”, antisense oligos with a cholesterol-moiety modification at the 3′, and present an optimized method to apply them onto 3D cultured embryonic pancreas. The method offers an approach to study the developmental functions of individual miRNAs and to evaluate miRNA targets, which is significantly faster and simpler than comparable genetics-based approaches.

Key words

Antagomir miRNA silencing Cholesterol conjugate Pancreas miRNA targets 



We thank Judith Maggenheim and Yuval Dor (Hadassah Medical School, The Hebrew University, Jerusalem, Israel) for protocols and advice and Tal Melkman-Zehavi for comments on this manuscript. EH is the incumbent of the Helen and Milton A Kimmelman Career Development Chair. This work was supported by grants from Juvenile Diabetes Research Foundation (#99-2007-71), the EFSD/D-Cure Young Investigator award, the Israel Science Foundation, the Yeda-Sela Center for Basic Research and the Wolfson Family Charitable Trust.


  1. 1.
    Lewis BP, Burge CB, Bartel DP. (2005). Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1), 15–20.PubMedCrossRefGoogle Scholar
  2. 2.
    Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2), 281–97.PubMedCrossRefGoogle Scholar
  3. 3.
    Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA. (1999). Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13(24), 3191–7.PubMedCrossRefGoogle Scholar
  4. 4.
    Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M. (2005). Silencing of microRNAs in vivo with “antagomirs”. Nature 438(7068), 685–9.PubMedCrossRefGoogle Scholar
  5. 5.
    Morton SU, Scherz PJ, Cordes KR, Ivey KN, Stainier DY, Srivastava D. (2008). microRNA-138 modulates cardiac patterning during embryonic development. Proc Natl Acad Sci USA 105(46), 17830–5.PubMedCrossRefGoogle Scholar
  6. 6.
    van Solingen C, Seghers L, Bijkerk R, Duijs JM, Roeten MK, van Oeveren-Rietdijk AM, Baelde HJ, Monge M, Vos JB, de Boer HC, Quax PH, Rabelink TJ, van Zonneveld AJ. (2008). Antagomir-Mediated Silencing of Endothelial Cell Specific MicroRNA-126 Impairs Ischemia-Induced Angiogenesis. J Cell Mol Med. 13(8A),1577–85.Google Scholar
  7. 7.
    Krützfeldt J, Kuwajima S, Braich R, Rajeev KG, Pena J, Tuschl T, Manoharan M, Stoffel M. (2007). Specificity, duplex degradation and subcellular localization of antagomirs. Nucleic Acids Res 35(9), 2885–92.PubMedCrossRefGoogle Scholar
  8. 8.
    Farh KK, Grimson A, Jan C, Lewis BP, Johnston WK, Lim LP, Burge CB, Bartel DP. (2005). The widespread impact of mammalian MicroRNAs on mRNA repression and evolution. Science 310(5755), 1817–21.PubMedCrossRefGoogle Scholar
  9. 9.
    Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. (2003). Prediction of mammalian microRNA targets. Cell 115(7), 787–98.PubMedCrossRefGoogle Scholar
  10. 10.
    Nolan T, Hands RE, Bustin SA. (2006). Quantification of mRNA using real-time RT-PCR. Nat Protoc 1(3), 1559–82.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  1. 1.Department of Molecular GeneticsWeizmann Institute of ScienceRehovotIsrael

Personalised recommendations