RLM-RACE, PPM-RACE, and qRT-PCR: An Integrated Strategy to Accurately Validate miRNA Target Genes

MicroRNAs (miRNAs) are important regulators involved in most biological processes in eukarya. They play critical roles in growth, development, signal transduction, or stress response by controlling gene expression at the posttranscriptional level. Identification and characterization of miRNA-targeted mRNAs is essential for the analysis of miRNA functions. In plants, the perfect complementarity between most miRNAs and their targets enables the accurate predictions of their targets, while slicing of the targeted mRNAs facilitates target validation through RNA Ligase-Mediated (RLM)-Rapid Amplification of cDNA Ends (RACE) method. However, this method only determines the 5'-end of the cleavage product. To more accurately validate the predicted target genes of miRNAs and exactly determine the cleavage sites within the targets, an integrated strategy comprising RLM-RACE, Poly(A) Polymerase-Mediated (PPM)-RACE, and qRT-PCR was developed. The efficiency of this method is illustrated by the precise sequence validation of predicted target mRNAs of miRNAs in grapevine, citrus, peach, and other fruit crops. Our on-going research indicates that RLM-RACE, PPM-RACE, and qRT-PCR are very effective in the verification of sequences of miRNA targets obtained by Degradome sequencing. The protocol for RLM-RACE, PPM-RACE, and qRT-PCR is rapid, effective, cheap, and can be completed within 2-3 days.