title: RNAi in primary human chondrocytes: efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression.
abstract: RNAi-mediated gene silencing is a recent, powerful tool 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 IkappaB degradation and NFkappaB 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 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.
authors.affiliation: Institute of Clinical Chemistry, University of Cologne, Kerpener Strasse 62, 50924 Köln, Germany. andreas.klatt@uk-koeln.de
authors.forename: Andreas R
authors.initials: AR
authors.lastname: Klatt
authors.forename: Gabriele
authors.initials: G
authors.lastname: Klinger
authors.forename: Dagmar
authors.initials: D
authors.lastname: Zech
authors.forename: Brigitte
authors.initials: B
authors.lastname: Paul-Klausch
authors.forename: Joerg H
authors.initials: JH
authors.lastname: Renno
authors.forename: Joachim
authors.initials: J
authors.lastname: Schmidt
authors.forename: Gebhart
authors.initials: G
authors.lastname: Malchau
authors.forename: Klaus
authors.initials: K
authors.lastname: Wielckens
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D019902:Chondrocytes / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D015850:Interleukin-6 / Q000096:biosynthesis; D007700:Kinetics; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D019892:eIF-2 Kinase / Q000096:biosynthesis; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D020672:I-kappa B Proteins; D015850:Interleukin-6; D020930:MAP Kinase Kinase Kinases; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D019892:eIF-2 Kinase; D000099151:MAP Kinase Kinase Kinase 7; C508600:IL6 protein, human; C439524:TNF protein, human