...
【24h】

A biochemical approach to identifying microRNA targets

机译:识别microRNA靶标的生化方法

获取原文
获取原文并翻译 | 示例
           

摘要

Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunopre-cipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway.
机译:识别microRNA(miRNA)的下游靶标对于了解细胞调控网络至关重要。我们为miRNA目标发现设计了一种直接的生化方法,该方法将RNA诱导的沉默复合物(RISC)纯化与结合的mRNA的微阵列分析相结合。因为已经分析了miR-124a的靶标,所以我们选择它作为模型。我们通过检查体外RISC与合成靶标RNA之间稳定结合的决定因素以及确定体内两个特征明确的靶标中miR-124a种子位点的阻遏和RISC共免疫沉淀的依赖性,来完善我们的方法。检查293细胞中miR-124靶标的完整谱图,既得到了一组在mRNA水平下调的蛋白(如先前观察到的),又得到了其mRNA水平不受miR-124a影响的一组蛋白。记者分析验证了这两种类型,扩大了可通过实验与miRNA途径相连的mRNA靶标的范围。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱:kefu@zhangqiaokeyan.com

京公网安备:11010802029741号 ICP备案号:京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有
  • 客服微信

  • 服务号