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A Method for Validating MicroRNAs in Plants by miR-RACE

  • Jinggui Fang
  • Xin Sun
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1287)

Abstract

miRNA prediction algorithms often fail to predict the accurate location of the mature miRNA in a precursor sequence with nucleotide-level precision. miRNAs-rapid amplification of cDNA ends (miR-RACE) is an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs). miR-RACE includes the following steps: miRNA-enriched library preparation, two specific 5′- and 3′-miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning. The most challenging step is the two gene-specific primers designed for the two RACE reactions. The miR-RACE protocol is rapid and can be executed and completed in 2–3 days.

Key words

Plant miRNAs Validation miR-RACE 

References

  1. 1.
    Reinhart BJ, Weinstein EG, Rhoades MW et al (2002) MicroRNAs in plants. Genes Dev 16:1616–1626CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Chapman EJ, Prokhnevsky AI, Gopinath K et al (2004) Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18:1179–1186CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Ramachandran V, Chen X (2008) Small RNA metabolism in Arabidopsis. Trends Plant Sci 13:368–374CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Grant-Downton R, Hafidh S, Twell D et al (2009) Small RNA pathways are present and functional in the angiosperm male gametophyte. Mol Plant 2:500–512CrossRefPubMedGoogle Scholar
  5. 5.
    Raman S, Greb T, Peaucelle A et al (2008) Interplay of miR164, CUP-SHAPED COTYLEDON genes and LATERAL SUPPRESSOR controls axillary meristem formation in Arabidopsis thaliana. Plant J 55:65–76CrossRefPubMedGoogle Scholar
  6. 6.
    Mathieu J, Yant LJ, Mürdter F et al (2009) Repression of flowering by the miR172 target SMZ. PLoS Biol 7:e10001CrossRefGoogle Scholar
  7. 7.
    Wang L, Mai YX, Zhang YC et al (2010) MicroRNA171c-targeted SCL6-II, SCL6-III, and SCL-IVgenes regulate shoot branching in Arabidopsis. Mol Plant 3:794–806CrossRefPubMedGoogle Scholar
  8. 8.
    Grigorova B, Mara C, Hollender C et al (2011) LEUNIG and SEUSS co-repressors regulate miR172 expression in Arabidopsis flowers. Development 138:2451–2456CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Thiebaut F, Rojas CA, Almeida KL et al (2012) Regulation of miR319 during cold stress in sugarcane. Plant Cell Environ 35:502–512CrossRefPubMedGoogle Scholar
  10. 10.
    Song CN, Fang JG, Wang C et al (2010) MiR-RACE, a new efficient approach to determine the precise sequences of computationally identified trifoliate orange (Poncirus trifoliata) MicroRNAs. PLoS ONE 5:e10861CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Wang C, Shangguan LF, Nicholas KK et al (2011) Characterization of microRNAs identified in a table grapevine cultivar with validation of computationally predicated grapevine miRNAs by miR-RACE. PLoS ONE 6:e21259CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Wang C, Han J, Liu C et al (2012) Identification of microRNAs from Amur grapes (Vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics. BMC Genomics 13:122CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Yu H, Song C, Jia Q et al (2011) Computational identification of microRNAs in apple expressed sequence tags and validation of their precise sequences by miR-RACE. Physiol Plant 144:56–70CrossRefGoogle Scholar
  14. 14.
    Zhang YP, Yu ML, Yu HP et al (2012) Computational identification of microRNAs in peach expressed sequence tags and validation of their precise sequences. Mol Biol Rep 39:1975–1987CrossRefPubMedGoogle Scholar
  15. 15.
    Zhang YP, Bai YH, Chen M et al (2013) Bioinformatics prediction of miRNAs in the Prunus persica genome with validation of their precise sequences by miR-RACE. Plant Physiol 170:80–92CrossRefGoogle Scholar
  16. 16.
    Dong QH, Han J, Yu HP et al (2012) Computational identification of microRNAs in strawberry expressed sequence tags and validation of their precise sequences by miR-RACE. J Hered 103:268–277CrossRefPubMedGoogle Scholar
  17. 17.
    Carter P (1986) Site-directed mutagenesis. Biochem J 237:1–7PubMedCentralPubMedGoogle Scholar
  18. 18.
    Peng RH, Xiong AS, Yao QH (2006) A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis. Appl Microbiol Biot 73:234–240CrossRefGoogle Scholar
  19. 19.
    Kovalic D, Kwak JH, Weisblum B (1991) General method for rapid direct cloning of DNA fragments generated by the polymerase chain reaction. Nucleic Acids Res 19:4560CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.College of HorticultureNanjing Agricultural UniversityNanjingPeople’s Republic of China

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