McClafferty Heather, Shipston Michael J
Centre for Discovery Brain Sciences, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK.
Methods Mol Biol. 2019;2009:151-168. doi: 10.1007/978-1-4939-9532-5_12.
The lack of specific pharmacological tools to interrogate the functional role of palmitoyl acyltransferases (zDHHCs) in mammalian cells has significantly hampered the understanding of this important gene family. Gene silencing by RNA interference (RNAi) is a process in eukaryotes that allows specific knockdown of the expression of proteins by targeting their coding mRNA. RNAi can thus be used as a proteomic tool to study the functional role of specific zDHHCs in cells by analyzing the effects of endogenous zDHHC knockdown on their protein targets or pathways. Here we describe the application of short interfering RNA (siRNA), a class of short (20-25 base pairs) double-stranded RNAs, to knockdown endogenous zDHHC enzymes expressed in human embryonic kidney (HEK293) cells and subsequent validation of knockdown efficiency using RT-qPCR to quantify zDHHC mRNA levels.
在哺乳动物细胞中，缺乏用于探究棕榈酰转移酶（zDHHCs）功能作用的特异性药理学工具，这严重阻碍了我们对这个重要基因家族的了解。RNA干扰（RNAi）介导的基因沉默是真核生物中的一个过程，它通过靶向蛋白质的编码mRNA来特异性降低蛋白质的表达。因此，RNAi可作为一种蛋白质组学工具，通过分析内源性zDHHC基因敲低对其蛋白质靶点或信号通路的影响，来研究特定zDHHCs在细胞中的功能作用。在此，我们描述了短干扰RNA（siRNA，一类短的（20 - 25个碱基对）双链RNA）在敲低人胚肾（HEK293）细胞中内源性zDHHC酶表达方面的应用，以及随后使用RT-qPCR定量zDHHC mRNA水平来验证敲低效率的过程。
