Tomasello Luisa, Cluts Landon, Croce Carlo M
Department of Cancer Biology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2019;1970:331-339. doi: 10.1007/978-1-4939-9207-2_18.
MicroRNAs (miRNAs) are small noncoding RNAs that are 22-25 nucleotides in length and control gene expression posttranscriptionally by degrading mRNAs or by translational repression. Many computational tools based on algorithms for target prediction have already been developed to find potential miRNA-target interactions. Since it is essential to confirm in silico predictions, experimental approaches have been improved to validate computationally predicted targets. One of the most widely used techniques is the luciferase assay which allows for the confirmation of specific binding between microRNA and the mRNA target using a reporter plasmid containing the 3' UTR of the target. Through the mutagenesis of this region it is possible to provide indirect evidence of the specific microRNA-mRNA interaction demonstrated using this assay. In this chapter we review the main experimental steps of the 3' UTR mutagenesis and the best way to apply this method to support and complete the luciferase assay procedure.
微小RNA（miRNA）是长度为22 - 25个核苷酸的小型非编码RNA，通过降解mRNA或翻译抑制在转录后控制基因表达。已经开发了许多基于算法的靶标预测计算工具来寻找潜在的miRNA - 靶标相互作用。由于在计算机上进行预测的确认至关重要，因此实验方法已得到改进以验证通过计算预测的靶标。最广泛使用的技术之一是荧光素酶测定法，该方法允许使用含有靶标3'非翻译区（UTR）的报告质粒来确认微小RNA与mRNA靶标之间的特异性结合。通过该区域的诱变，可以为使用该测定法证明的特定微小RNA - mRNA相互作用提供间接证据。在本章中，我们回顾了3'UTR诱变的主要实验步骤以及应用该方法来支持和完成荧光素酶测定程序的最佳方法。
