Wang Chen, Fang Jinggui
College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China.
Methods Mol Biol. 2015;1296:175-86. doi: 10.1007/978-1-4939-2547-6_16.
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.
微小RNA（miRNA）是参与真核生物大多数生物学过程的重要调节因子。它们通过在转录后水平控制基因表达，在生长、发育、信号转导或应激反应中发挥关键作用。鉴定和表征miRNA靶向的mRNA对于分析miRNA功能至关重要。在植物中，大多数miRNA与其靶标之间的完美互补性使得能够准确预测其靶标，而靶向mRNA的切割则通过RNA连接酶介导（RLM）的cDNA末端快速扩增（RACE）方法促进靶标验证。然而，该方法仅确定切割产物的5'末端。为了更准确地验证miRNA的预测靶基因并精确确定靶标内的切割位点，开发了一种包括RLM-RACE、聚腺苷酸聚合酶介导（PPM）-RACE和qRT-PCR的综合策略。葡萄、柑橘、桃子和其他水果作物中miRNA预测靶标mRNA的精确序列验证说明了该方法的有效性。我们正在进行的研究表明，RLM-RACE、PPM-RACE和qRT-PCR在验证通过降解组测序获得的miRNA靶标序列方面非常有效。RLM-RACE、PPM-RACE和qRT-PCR的方案快速、有效、廉价，并且可以在2-3天内完成。
