RNAi in primary human chondrocytes: Efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression
RNAi in primary human chondrocytes: Efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression
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DOI：
10.1016/j.biologicals.2006.12.003
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发表时间：
2007-10-01
期刊：
影响因子：
1.7
通讯作者：
Wielckens, Klaus
中科院分区：
文献类型：
--
作者：
Klatt, Andreas R.;Klinger, Gabriele;Wielckens, Klaus
RNA干扰介导的基因沉默是一个新的、强有力的研究基因功能的工具。控制实验因素如转染效率、siRNA浓度、基因抑制水平、基因抑制动力学或非特异性效应是获得稳健结果的关键。在这篇方法论文中，我们比较了不同转染试剂在原代人软骨细胞（PHCs）中的效率。我们研究了TAK 1基因抑制效率和动力学的mRNA和蛋白质水平上取决于所使用的siRNA浓度。此外，我们评估了PKR、IL-6和TNF-α诱导，以及I κ B降解和NF κ B活化作为非特异性siRNA作用的对照参数。PKR和IL-6被证明是siRNA转染引起的细胞炎症反应的适当标志物。此外，我们比较了不同siRNA（沉默、非沉默、经典21-mer和25-mer stealth（TM）siRNA）诱导细胞炎症反应的能力。我们发现在PHCs中细胞反应的发生是所使用的特定siRNA序列的功能。因此，在任何功能基因验证之前，分析和比较基因沉默siRNA和对照siRNA的脱靶效应是至关重要的。(C)2007年，国际生物制品协会。由爱思唯尔有限公司出版。保留所有权利。
RNAi-mediated gene silencing is a recent, powerful too] to investigate gene function. Controlling for experimental factors such as transfection efficiencies, siRNA concentration, gene suppression levels, gene suppression kinetics, or non-specific effects is key to robust results. In this methods paper, we compare the efficiencies of different transfection reagents in primary human chondrocytes (PHCs). We investigated TAK1 gene suppression efficiencies and kinetics on the mRNA and protein level depending on the siRNA concentration used. Furthermore, we evaluated PKR, IL-6, and TNF-alpha induction, as well as I kappa B degradation and NF kappa B activation as control parameters of non-specific siRNA effects. PKR and IL-6 proved to be appropriate markers of cellular inflammatory responses resulting from siRNA transfection. In addition, we compared different siRNAs (silencing, non-silencing, classic 21-mer, and 25-mer stealth (TM) siRNA) with respect to their capacity to induce cellular inflammatory responses. We found the occurrence of cellular responses in PHCs to be a function of the specific siRNA sequence in use. Hence, it is essential to analyze and to compare gene silencing siRNAs and control siRNAs with respect to their off-target effects prior to any functional gene validation. (C) 2007 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.