MicroRNAs (miRNAs) are small noncoding RNAs of 22–25 nucleotides that control gene expression at the posttranscriptional level through the degradation of mRNAs or translational repression. In the last 15 years, the study of these small molecules helped elucidate their role in the regulation of many cellular processes and the onset and development of several diseases. Therefore, many computational tools based on algorithms for target prediction have been developed to identify potential miRNA–target interactions. The improvement of experimental approaches to more easily and quickly confirm in silico predictions has become essential for the study of these small RNAs and their molecular functions. In this chapter, we summarized the principal steps of one of the most used techniques for the validation of microRNA targets, the Luciferase assay, thus explaining the underlying principles and the procedures to apply it best.
MicroRNA target validation Luciferase assay Cloning In vitro techniques
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
Janga SC, Vallabhaneni S (2011) MicroRNAs as post-transcriptional machines and their interplay with cellular networks. Adv Exp Med Biol 722:59–74CrossRefGoogle Scholar
Solberg N, Krauss S (2013) Luciferase assay to study the activity of a cloned promoter DNA fragment. Methods Mol Biol 977:65–78CrossRefGoogle Scholar
Zhang S, Cahalan MD (2007) Purifying plasmid DNA from bacterial colonies using the Qiagen Miniprep kit. J Vis Exp 6:247Google Scholar
Cranenburgh RM (2004) An equation for calculating the volumetric ratios required in a ligation reaction. Appl Microbiol Biotechnol 65:200–202CrossRefGoogle Scholar