Experimental Validation of MicroRNA Targets Using a Luciferase Reporter System
Experimental Validation of MicroRNA Targets Using a Luciferase Reporter System
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DOI：
10.1007/978-1-61779-083-6_11
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发表时间：
2011-01-01
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影响因子：
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通讯作者：
Nicolas, Francisco E.
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文献类型：
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作者：
Nicolas, Francisco E.
microRNAs（miRNAs）是一类小的非编码转录本，通过与靶信使RNA（mRNAs）配对抑制基因表达。人类基因组编码数百种不同的miRNA，并且预测它们靶向参与多种生理过程如发育和细胞身份的数千种mRNA。在动物中，mRNA靶标的鉴定是复杂的，因为大多数miRNA及其靶mRNA不具有精确或几乎精确的互补性。动物miRNA以不完全序列同源性结合其mRNA靶标的这种倾向代表了鉴定miRNA靶标的相当大的挑战。基于保守性的计算算法和基于表达谱的实验方法充斥着包含大量假阳性和假阴性预测的候选基因列表以及覆盖每个miRNA的真实的直接靶点列表的间接靶点。目前，唯一可用的工具来验证序列作为miRNA的直接靶标是基于携带候选序列的报告基因的系统。在此，详细描述了基于荧光素酶蛋白产生的发光的miRNA靶标验证报告基因系统，包括报告基因构建体的设计、其在模型细胞系中的表达以及其使用荧光素酶测定的测量。
MicroRNAs (miRNAs) are a class of small noncoding transcripts that repress gene expression by pairing with their target messenger RNAs (mRNAs). The human genome codes for hundreds of different miRNAs and it is predicted that they target thousands of mRNAs involved in a wide variety of physiological processes such as development and cell identity. In animals, the identification of mRNA targets is complex because most miRNAs and their target mRNAs do not have exact or nearly exact complementarity. This tendency of animal miRNAs to bind their mRNA targets with imperfect sequence homology represents a considerable challenge to identifying miRNA targets. Computational algorithms based on conservation and experimental approaches based on expression profiles are flooding the literature with lists of candidate genes containing a large number of false-positive and false-negative predictions and indirect targets that cover the real list of direct targets for each miRNA. Currently, the only available tools to validate a sequence as a direct target of an miRNA are the systems based on a reporter gene carrying the candidate sequence. Here, an miRNA target validation reporter gene system based on the luminescence generated by the luciferase protein is described in detail, including the design of the reporter constructs, its expression in a model cell line and its measurement using a luciferase assay.