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Target Validation of Plant microRNAs

  • César LlaveEmail author
  • José Manuel Franco-Zorrilla
  • Roberto Solano
  • Daniel Barajas
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 732)

Abstract

microRNAs (miRNAs) regulate gene expression through sequence-specific interactions with cognate mRNAs that result in translational inhibition, mRNA decay, or slicing within the region of complementarity. miRNA processing activity on complementary target mRNAs generates 3′ end cleavage products that contain ­ligation-competent, 5′-monophosphates. Precise mapping of miRNA-directed cleavage sites within target transcripts is, therefore, possible using RNA ligase-mediated 5′ amplification of cDNA ends (RLM-RACE). Here, we provide a comprehensive RLM-RACE-based protocol for the amplification of 5′ ends derived from cleaved transcripts resulting from miRNA-guided cleavage events. Novel strategies for high-throughput analysis of miRNA cleavage products have emerged as powerful tools for the novo identification of miRNA targets in a genomic perspective. In this work, we also describe a novel methodology for genome-wide identification of miRNA targets that exploits RLM-RACE for non-sequence-specific enrichment of cleaved transcripts, T7 RNA polymerase-mediated amplification of target products, and microarray hybridization.

Key words

RLM-RACE microRNAs microRNA targets Gene regulation Microarray hybridization 

Notes

Acknowledgments

This work was supported by grants to CL from the Spanish Ministry of Science and Technology (GEN2003-20222-CO2-00 and BIO2006-13107), and Comunidad de Madrid (Spain) (CCG07-CSIC/GEN-1804), and by grants to RS from the Ministry of Science and Technology (BIO2004-02502, BIO2007-66935, GEN2003-20218-C02-02 and CSD2007-00057-B). J.M.F.-Z. is supported by a contract I3P-Doctores (CSIC).

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Copyright information

© Humana Press 2011

Authors and Affiliations

  • César Llave
    • 1
    Email author
  • José Manuel Franco-Zorrilla
  • Roberto Solano
  • Daniel Barajas
  1. 1.Department of Environmental BiologyCentro de Investigaciones Biológicas – CSICMadridSpain

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