Cheng Angie, Magdaleno Susan, Vlassov Alexander V
Molecular and Cell Biology Division, Life Technologies, Austin, TX 78744-1832, USA.
Methods Mol Biol. 2011;764:199-213. doi: 10.1007/978-1-61779-188-8_13.
RNA interference (RNAi) is a mechanism by which the introduction of small interfering RNAs (siRNAs) into cultured cells causes degradation of the complementary mRNA. Applications of RNAi include gene function analysis, pathway analysis, and target validation. While RNAi experiments have become common practice in research labs, multiple factors can influence the extent of siRNA-induced knockdown (and thus biological outcome). A properly designed and selected siRNA sequence, siRNA modification format, choice of transfection reagent/technique, optimized protocols of siRNA in vitro delivery, and an appropriate and optimized readout are all critical for ensuring a successful experiment. In this chapter, we describe a typical in vitro siRNA experiment with optimization of transfection conditions and analysis of siRNA potency, i.e., mRNA knockdown with quantitative real-time PCR.
RNA干扰（RNAi）是一种机制，通过将小干扰RNA（siRNA）导入培养细胞，可导致互补mRNA的降解。RNAi的应用包括基因功能分析、信号通路分析和靶点验证。虽然RNAi实验已成为研究实验室的常规操作，但多种因素会影响siRNA诱导的基因敲低程度（进而影响生物学结果）。设计和选择合适的siRNA序列、siRNA修饰形式、转染试剂/技术的选择、优化的siRNA体外递送方案以及适当且优化的检测指标，对于确保实验成功都至关重要。在本章中，我们描述了一个典型的体外siRNA实验，包括转染条件的优化和siRNA效力分析，即通过定量实时PCR检测mRNA敲低情况。
