Western blot evaluation of siRNA delivery by pH-responsive peptides.
Western blot evaluation of siRNA delivery by pH-responsive peptides.
复制标题
DOI：
10.1007/978-1-62703-311-4_5
复制
发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Lam, Jenny K W
中科院分区：
文献类型：
--
作者：
Liang, Wanling;Mason, A James;Lam, Jenny K W
通过使用小干扰RNA (siRNA) 的RNA 干扰(RNAi) 技术进行基因沉默，已发展成为药物发现中靶标识别和验证的重要工具，并具有巨大的治疗潜力。然而，有效递送到细胞中对 siRNA 的使用提出了重大挑战。近年来，pH 响应性细胞穿透肽作为递送载体引起了相当大的关注，因为它们能够跨生物膜运输其货物和/或促进内体逃逸并防止溶酶体降解。为了评估基于 pH 响应肽的 siRNA 递送系统的体外转染效率，通常采用蛋白质印迹技术。该方法通过分析样品中的目的蛋白，以最少的设备要求，提供了一种简单、高效且经济的方法来研究 siRNA 的基因沉默效应。本章介绍了通过蛋白质印迹实验方案进行 siRNA 递送和分析，以定性和定量评估基因沉默效率和选择性。
Gene silencing, via RNA interference (RNAi) technologies using small interfering RNA (siRNA), has been developed as an important tool for target identification and validation in drug discovery and has huge therapeutic potential. However, effective delivery into cells presents a major challenge to the use of siRNA. pH responsive cell-penetrating peptides have attracted considerable attention in recent years as delivery vectors due to their ability to transport their cargos across the biological membrane and/or to promote endosomal escape and prevent lyososomal degradation. To evaluate the in vitro transfection efficiency of the pH responsive peptide-based siRNA delivery system, the western blotting technique is commonly employed. This method offers a simple, efficient and economical way to study the gene silencing effect of the siRNA by analysing the protein of interest in a sample with minimum equipment requirement. This chapter provides a description of siRNA delivery and analysis by western blotting protocols for qualitatively and quantitatively assessing gene silencing efficiency and selectivity.