Dual Luciferase Gene Reporter Assays to Study miRNA Function
Dual Luciferase Gene Reporter Assays to Study miRNA Function
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DOI：
10.1007/978-1-4939-2547-6_17
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发表时间：
2015-01-01
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通讯作者：
Rederstorff, Mathieu
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文献类型：
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作者：
Clement, Thomas;Salone, Veronique;Rederstorff, Mathieu
本章描述了一种最可靠的定量分析方法，用于测试特定miRNA使用荧光素酶报告基因沉默可能的靶基因。实验程序首先包括克隆萤火虫荧光素酶报告基因下游miRNA预测mRNA靶标的3'UTR的野生型和突变形式。接下来，将每个构建体与miRNA一起共转染到HeLa细胞中，并监测报告基因的表达。荧光素酶水平的变化将指示miRNA是否可以结合UTR并调节其表达。
This chapter describes one of the most reliable quantitative assays to test the silencing of a possible target gene by a specific miRNA using a luciferase reporter gene. The experimental procedure first consists in cloning both the wild-type and mutated forms of the 3'UTR of the miRNA predicted mRNA target downstream of a firefly luciferase reporter. Next, each construct is co-transfected together with the miRNA into HeLa cells, and the reporter expression is monitored. Changes in luciferase levels will indicate whether or not a miRNA can bind to the UTR and regulate its expression.