Molecular Biology Reports

, Volume 39, Issue 2, pp 1783–1790 | Cite as

Isolation and cloning of microRNAs from recalcitrant plant tissues with small amounts of total RNA: a step-by step approach

  • Chee Wei Yew
  • S. Vijay Kumar


MicroRNAs (miRNAs) are small RNAs (sRNAs) with approximately 21–24 nucleotides in length. They regulate the expression of target genes through the mechanism of RNA silencing. Conventional isolation and cloning of miRNAs methods are usually technical demanding and inefficient. These limitations include the requirement for high amounts of starting total RNA, inefficient ligation of linkers, high amount of PCR artifacts and bias in the formation of short miRNA-concatamers. Here we describe in detail a method that uses 80 μg of total RNA as the starting material. Enhancement of the ligation of sRNAs and linkers with the use of polyethylene glycol (PEG8000) was described. PCR artifacts from the amplification of reverse-transcribed sRNAs were greatly decreased by using lower concentrations of primers and reducing the number of amplification cycles. Large concatamers with up to 1 kb in size with around 20 sRNAs/concatamer were obtained by using an optimized reaction condition. This protocol provide researchers with a rapid, efficient and cost-effective method for the construction of miRNA profiles from plant tissues containing low amounts of total RNA, such as fruit flesh and senescent leaves.


microRNA Fruits Small RNA library Concatamerization RNAi 



The authors thank the Ministry of Higher Education, Malaysia, for supporting this work through the Fundamental Research Grant Scheme (Grant no. FRG164-SG-2008).

Conflicts of interest

The authors declare that they have no competing interests.


  1. 1.
    Yang T, Xue L, An L (2007) Functional diversity of miRNA in plants. Plant Sci 172:423–432CrossRefGoogle Scholar
  2. 2.
    Chuck G, Candela H, Hake S (2008) Big impacts by small RNAs in plant development. Curr Opin Plant Biol 12:1–6Google Scholar
  3. 3.
    Sunkar R, Zhu J (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019PubMedCrossRefGoogle Scholar
  4. 4.
    Sunkar R, Girke T, Jain PK, Zhu J (2005) Cloning and characterization of microRNAs from rice. Plant Cell 17:1397–1411PubMedCrossRefGoogle Scholar
  5. 5.
    Itaya A, Bundschuh R, Archual AJ, Joung J, Fei Z, Dai X, Zhao PX, Tang Y, Nelson RS, Ding B (2008) Small RNAs in tomato fruit and leaf development. Biochim Biophys Acta 1779:99–107PubMedGoogle Scholar
  6. 6.
    Zhang BH, Pan XP, Wang QL, Cobb GB, Anderson TA (2007) Identification and characterization of new plant microRNAs using EST analysis. Cell Res 15:336–360CrossRefGoogle Scholar
  7. 7.
    Sunkar R, Jagadeeswaran G (2008) In silico identification of conserved microRNAs in large number of diverse plant species. BMC Plant Biol 8:37–49PubMedCrossRefGoogle Scholar
  8. 8.
    Arazi T, Talmor-Neiman M, Stav R, Riese M, Huijser P, Baulcombe D (2005) Cloning and characterization of microRNAs from moss. Plant J 43:837–848PubMedCrossRefGoogle Scholar
  9. 9.
    Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862PubMedCrossRefGoogle Scholar
  10. 10.
    Carra A, Gambino G, Schubert A (2007) A cetyltrimethylammonium bromide-based method to extract low-molecular-weight RNA from polysaccharide-rich plant tissues. Anal Biochem 360:318–320PubMedCrossRefGoogle Scholar
  11. 11.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297PubMedCrossRefGoogle Scholar
  12. 12.
    Aravin A, Tuschl T (2005) Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett 579:5830–5840PubMedCrossRefGoogle Scholar
  13. 13.
    Ho CK, Wang LK, Lima CD, Shuman S (2004) Structure and mechanism of RNA ligase. Structure 12:327–339PubMedGoogle Scholar
  14. 14.
    Harrison B, Zimmerman S (1984) Polymer-stimulated ligation: enhanced ligation of oligo- and polynucleotides by T4 RNA ligase in polymer solutions. Nucleic Acids Res 12:8235–8251PubMedCrossRefGoogle Scholar
  15. 15.
    Tessier DC, Brousseau R, Vernet T (1986) Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase. Anal Biochem 158:171–178PubMedCrossRefGoogle Scholar
  16. 16.
    Lu C, Meyers BC, Green PJ (2007) Construction of small RNA cDNA libraries for deep sequencing. Methods 43:110–117PubMedCrossRefGoogle Scholar
  17. 17.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415PubMedCrossRefGoogle Scholar
  18. 18.
    Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL, Cao X, Carrington JC, Chen X, Green PJ, Griffiths-Jones S, Jacobsen SE, Mallory AC, Martienssen RA, Poethig RS, Qi Y, Vaucheret H, Voinnet O, Watanabe Y, Weigel D, Zhu J (2008) Criteria for annotation of plant microRNAs. Plant Cell 20:3186–3190PubMedCrossRefGoogle Scholar
  19. 19.
    Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D (2005) Specific effects of microRNAs on the plant transcriptome. Dev Cell 8:517–527PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Biotechnology Research InstituteUniversiti Malaysia SabahKota KinabaluMalaysia

Personalised recommendations