In Situ Detection of Mature miRNAs in Plants Using LNA-Modified DNA Probes

  • Xiaozhen Yao
  • Hai Huang
  • Lin XuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 883)


MicroRNAs (miRNAs) play important roles in development in plants, and some miRNAs show developmentally regulated organ- and tissue-specific expression patterns. Therefore, in situ detection of mature miRNAs is important for understanding the functions of both miRNAs and their targets. The construction of promoter–reporter fusions and examination of their in planta expression have been widely used and the results obtained thus far are rather informative; however, in some cases, the length of promoter that contains the entire regulatory elements is difficult to determine. In addition, traditional in situ hybridization with the antisense RNA fragment as the probe usually fails to detect miRNAs because the mature miRNAs are too short (∼21 nt) to exhibit stable hybridization signals. In recent years, the locked nucleic acid (LNA)-modified DNA probe has been successfully used in animals and plants to detect small RNAs. Here, we describe a modified protocol using LNA-modified DNA probes to detect mature miRNAs in plant ­tissues, including the design of LNA probes and detailed steps for the in situ hybridization experiment, using Arabidopsis miR165 as an example.

Key words

Plant miRNA In situ hybridization LNA-modified DNA probe 



We are grateful to H. Wang for discussion with the manuscript. We thank Dr. Y. Eshed and Dr. I. Pekker for sharing experience on LNA probes. This work was supported by grants from the Chief Scientist Program of Shanghai Institutes for Biological Sciences, the Chinese National Scientific Foundation 30630041, and Chinese Academy of Sciences (KSCX2-YW-N-057).


  1. 1.
    Bartel DP (2004) MicroRNAs: genomics, ­biogenesis, mechanism, and function. Cell 116:281–297PubMedCrossRefGoogle Scholar
  2. 2.
    Wienholds E, Plasterk RH (2005) MicroRNA function in animal development. FEBS Lett 579:5911–5922PubMedCrossRefGoogle Scholar
  3. 3.
    Wu L, Zhou H, Zhang Q, Zhang J, Ni F, Liu C, Qi Y (2010) DNA methylation mediated by a microRNA pathway. Mol Cell 38:465–475PubMedCrossRefGoogle Scholar
  4. 4.
    Chellappan P, Xia J, Zhou X, Gao S, Zhang X, Coutino G, Vazquez F, Zhang W, Jin H (2010) siRNAs from miRNA sites mediate DNA methylation of target genes. Nucleic Acids Res 38:6883–6894PubMedCrossRefGoogle Scholar
  5. 5.
    Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403:901–906PubMedCrossRefGoogle Scholar
  6. 6.
    Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 15:2730–2741PubMedCrossRefGoogle Scholar
  7. 7.
    Mallory AC, Bartel DP, Bartel B (2005) MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17:1360–1375PubMedCrossRefGoogle Scholar
  8. 8.
    Yang L, Liu Z, Lu F, Dong A, Huang H (2006) SERRATE is a novel nuclear regulator in primary microRNA processing in Arabidopsis. Plant J 47:841–850PubMedCrossRefGoogle Scholar
  9. 9.
    Diederichs S, Haber DA (2007) Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression. Cell 131:1097–1108PubMedCrossRefGoogle Scholar
  10. 10.
    Parizotto EA, Dunoyer P, Rahm N, Himber C, Voinnet O (2004) In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev 18:2237–2242PubMedCrossRefGoogle Scholar
  11. 11.
    Wang JW, Wang LJ, Mao YB, Cai WJ, Xue HW, Chen XY (2005) Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis. Plant Cell 17:2204–2216PubMedCrossRefGoogle Scholar
  12. 12.
    Jung JH, Park CM (2007) MIR166/165 genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in Arabidopsis. Planta 225:1327–1338PubMedCrossRefGoogle Scholar
  13. 13.
    Raman S, Greb T, Peaucelle A, Blein T, Laufs P, Theres K (2008) Interplay of miR164, CUP-SHAPED COTYLEDON genes and LATERAL SUPPRESSOR controls axillary meristem formation in Arabidopsis thaliana. Plant J 55:65–76PubMedCrossRefGoogle Scholar
  14. 14.
    Gutierrez L, Bussell JD, Pacurar DI, Schwambach J, Pacurar M, Bellini C (2009) Phenotypic plasticity of adventitious rooting in Arabidopsis is controlled by complex regulation of AUXIN RESPONSE FACTOR transcripts and microRNA abundance. Plant Cell 21:3119–3132PubMedCrossRefGoogle Scholar
  15. 15.
    Yao X, Wang H, Li H, Yuan Z, Li F, Yang L, Huang H (2009) Two types of cis-acting elements control the abaxial epidermis-specific transcription of the MIR165a and MIR166a genes. FEBS Lett 583:3711–3717PubMedCrossRefGoogle Scholar
  16. 16.
    Yoon EK, Yang JH, Lim J, Kim SH, Kim SK, Lee WS (2010) Auxin regulation of the microRNA390-dependent transacting small interfering RNA pathway in Arabidopsis lateral root development. Nucleic Acids Res 38:1382–1391PubMedCrossRefGoogle Scholar
  17. 17.
    Rodriguez JB, Marquez VE, Nicklaus MC, Mitsuya H, Barchi JJ Jr (1994) Conformationally locked nucleoside analogues. Synthesis of dideoxycarbocyclic nucleoside analogues structurally related to neplanocin C. J Med Chem 37:3389–3399PubMedCrossRefGoogle Scholar
  18. 18.
    Braasch DA, Corey DR (2001) Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem Biol 8:1–7PubMedCrossRefGoogle Scholar
  19. 19.
    Wengel J, Petersen M, Nielsen KE, Jensen GA, Hakansson AE, Kumar R, Sorensen MD, Rajwanshi VK, Bryld T, Jacobsen JP (2001) LNA (locked nucleic acid) and the diastereoisomeric alpha-L-LNA: conformational tuning and high-affinity recognition of DNA/RNA targets. Nucleosides Nucleotides Nucleic Acids 20:389–396PubMedCrossRefGoogle Scholar
  20. 20.
    Hakansson AE, Wengel J (2001) The adenine derivative of alpha-L-LNA (alpha-L-ribo ­configured locked nucleic acid): synthesis and high-affinity hybridization towards DNA, RNA, LNA and alpha-L-LNA complementary sequences. Bioorg Med Chem Lett 11:935–938PubMedCrossRefGoogle Scholar
  21. 21.
    Randazzo A, Esposito V, Ohlenschlager O, Ramachandran R, Virgilio A, Mayol L (2005) Structural studies on LNA quadruplexes. Nucleosides Nucleotides Nucleic Acids 24:795–800PubMedCrossRefGoogle Scholar
  22. 22.
    Valoczi A, Hornyik C, Varga N, Burgyan J, Kauppinen S, Havelda Z (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 32:e175PubMedCrossRefGoogle Scholar
  23. 23.
    Wienholds E, Kloosterman WP, Miska E, Alvarez-Saavedra E, Berezikov E, de Bruijn E, Horvitz HR, Kauppinen S, Plasterk RH (2005) MicroRNA expression in zebrafish embryonic development. Science 309:310–311PubMedCrossRefGoogle Scholar
  24. 24.
    Kloosterman WP, Wienholds E, de Bruijn E, Kauppinen S, Plasterk RH (2006) In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods 3:27–29PubMedCrossRefGoogle Scholar
  25. 25.
    Valoczi A, Varallyay E, Kauppinen S, Burgyan J, Havelda Z (2006) Spatio-temporal accumulation of microRNAs is highly coordinated in developing plant tissues. Plant J 47:140–151PubMedCrossRefGoogle Scholar
  26. 26.
    Long JA, Moan EI, Medford JI, Barton MK (1996) A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379:66–69PubMedCrossRefGoogle Scholar
  27. 27.
    You Y, Moreira BG, Behlke MA, Owczarzy R (2006) Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res 34:e60PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.National Laboratory of Plant Molecular GeneticsShanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of SciencesShanghaiChina

Personalised recommendations