Cheng Angie, Vlassov Alexander V, Magdaleno Susan
Molecular and Cell Biology Division, Life Technologies, Austin, TX, USA.
Methods Mol Biol. 2011;764:183-97. doi: 10.1007/978-1-61779-188-8_12.
RNA interference (RNAi) is a regulatory mechanism of eukaryotic cells that uses small interfering RNAs (siRNA) to direct homology-dependent control of gene activity. Applications of RNAi include functional genomics, in vivo target validation, and gene-specific medicines. A key to in vivo application of siRNA is the advancement of efficient delivery to organs, tissues, or cell types of interest. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (post-transfection) and in animals (post- injection). We have adopted the Applied Biosystems TaqMan(®) based stem-loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these needs. In this chapter, application protocols are described, which enable robust quantification of siRNA, including chemically modified molecules, in vitro and in vivo.
RNA干扰（RNAi）是真核细胞的一种调控机制，它利用小干扰RNA（siRNA）来指导对基因活性的同源依赖性控制。RNAi的应用包括功能基因组学、体内靶点验证和基因特异性药物。siRNA在体内应用的一个关键是提高向感兴趣的器官、组织或细胞类型的有效递送。需要开发可靠且易于使用的检测方法来评估siRNA的递送效率和分布，研究siRNA在细胞中（转染后）和动物体内（注射后）的途径及稳定性。我们采用了最初为定量细胞内源性微小RNA而开发的基于Applied Biosystems TaqMan®的茎环RT-PCR技术来满足这些需求。在本章中，将描述应用方案，这些方案能够在体外和体内对包括化学修饰分子在内的siRNA进行可靠定量。
