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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
Article

Abstract

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.

Keywords

microRNA Fruits Small RNA library Concatamerization RNAi 

Notes

Acknowledgments

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.

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Biotechnology Research InstituteUniversiti Malaysia SabahKota KinabaluMalaysia

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