Rice Protocols pp 109-118 | Cite as

Cloning of Small RNAs for the Discovery of Novel MicroRNAs in Plants

  • Guru Jagadeeswaran
  • Ramanjulu SunkarEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 956)


Endogenous small RNAs can be grouped into several distinct classes of 21-nt-long microRNAs (miRNAs), short interfering RNAs (siRNAs), trans-acting siRNAs (tasiRNAs), and 24-nt long heterochromatic siRNAs. miRNAs are increasingly being recognized as significant effectors of gene regulation in a wide range of organisms. These molecules are typically ∼21-nt long and are amenable for cloning by streamlined protocols. Here we detail the methodology for cloning small RNAs in rice to identify novel miRNAs and other important small RNAs. Briefly, small RNA molecules are size fractionated, attached to adaptors, and subsequently converted into cDNA and PCR amplified. Current high-throughput sequencing technologies allow sequencing of the PCR products directly.

Key words

MciroRNAs Small RNAs Posttranscriptional gene regulation Rice 



This research was supported by the Oklahoma Agricultural Experiment Station and the USDA/NRI grant (2007-02019).


  1. 1.
    Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53PubMedCrossRefGoogle Scholar
  2. 2.
    Mallory AC, Vaucheret H (2006) Functions of microRNAs and related small RNAs in plants. Nat Genet 38 Suppl: S31–S36Google Scholar
  3. 3.
    Sunkar R, Chinnusamy V, Zhu J, Zhu JK (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12:301–309PubMedCrossRefGoogle Scholar
  4. 4.
    Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136:669–687PubMedCrossRefGoogle Scholar
  5. 5.
    Katiyar-Agarwal S, Gao S, Vivian-Smith A, Jin H (2007) A novel class of bacteria-induced small RNAs in Arabidopsis. Genes Dev 21:3123–3134PubMedCrossRefGoogle Scholar
  6. 6.
    Lu S, Sun YH, Chiang VL (2008) Stress-responsive microRNAs3 in Populus. Plant J 55:131–151PubMedCrossRefGoogle Scholar
  7. 7.
    Ruiz-Ferrer V, Voinnet O (2009) Roles of plant small RNAs in biotic stress responses. Annu Rev Plant Biol 60:485–510PubMedCrossRefGoogle Scholar
  8. 8.
    Sunkar R, Girke T, Jain PK, Zhu JK (2005) Cloning and characterization of microRNAs from rice. Plant Cell 17:1397–1411PubMedCrossRefGoogle Scholar
  9. 9.
    Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes Dev 19:517–529PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyOklahoma State UniversityStillwaterUSA

Personalised recommendations