Comparing two approaches of miR-34a target identification, biotinylated-miRNA pulldown vs miRNA overexpression
Comparing two approaches of miR-34a target identification, biotinylated-miRNA pulldown vs miRNA overexpression
复制标题
比较两种 miR-34a 靶标识别方法：生物素化 miRNA 下拉与 miRNA 过表达
DOI：
10.1080/15476286.2017.1391441
复制
发表时间：
2018-01-01
期刊：
影响因子：
4.1
通讯作者：
Shan, Ge
中科院分区：
文献类型：
--
作者：
Awan, Hassaan Mehboob;Shah, Abdullah;Shan, Ge
微小RNA（miRNAs）是基因表达的关键调节因子。为了阐明miRNAs的功能作用，鉴定它们的直接靶标至关重要。关于生物素化的miRNA模拟物的下拉是否可用于鉴定miRNA靶标存在争议。在此我们表明，生物素标记的miR - 34a可被加载到AGO2上，并且AGO2免疫沉淀能够下拉生物素化的miR - 34a（生物素 - miR下拉）。对生物素 - miR下拉的RNA进行的RNA测序（RNA - seq）有效地鉴定了miR - 34a的mRNA靶标，这可通过荧光素酶测定法进行验证。与生物素 - miR下拉方法相反，miR - 34a过表达样本的RNA - seq在鉴定miR - 34a的直接靶标方面价值有限。似乎3' - 生物素标记的miRNA的下拉至少对于miR34a而言能够鉴定真正的微小RNA靶标。
ABSTRACT microRNAs (miRNAs) are critical regulators of gene expression. For elucidating functional roles of miRNAs, it is critical to identify their direct targets. There are debates about whether pulldown of biotinylated miRNA mimics can be used to identify miRNA targets or not. Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays. In contrast to the approach of Bio-miR pulldown, RNA-seq of miR-34a overexpression samples had limited value in identifying direct targets of miR-34a. It seems that pulldown of 3′-Biotin-tagged miRNA can identify bona fide microRNA targets at least for miR-34a.