title: [Comparison of efficiency and cytotoxicity of different transfection reagents in transfecting RIP140-siRNA into Kupffer cells].
abstract: OBJECTIVE
  To compare the efficiency and cytotoxicity of different transfection reagents used in transfection of RIP140-siRNA into Kupffer cells to optimize the transfection conditions.
  
  
  METHODS
  Kupffer cells were transfected with RIP140-siRNA labeled with GFP as the reporter gene using lipofectamine 2000, Roche reagent (X-treme GENE siRNA Transfection Reagent) and puro screening lentivirus (1.0×10(8) TU/mL) as the transfection reagents. The transfection effect was observed under a fluorescent inverted microscope, and laser scanning confocal microscopy was used to analyze RIP140 expression in trasnfected Kupffer cells. Flow cytometry was performed to detect cell apoptosis, and CCK-8 test was used to evaluate the cell proliferation inhibition. RT-RCR and Western blotting were performed to detect the expressions of RIP140 mRNA and protein in the trasnfected cells.
  
  
  RESULTS
  Puro screening lentivirus yielded the highest cell transfection efficiency, which exceeded 90%, followed by Roche reagent and then by lipofectamine 2000. Flow cytometry and CCK-8 test showed that the cytotoxicity was the mildest with Roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. The cells trasnfected with lentivirus showed a significantly lower RIP140 expression than cells trasnfected with lipofectamine 2000 and Roche reagent (P<0.05).
  
  
  CONCLUSIONS
  In Kupffer cells, lentivirus-mediated transfection, as compared with the other two trasnfection reagents, can achieve good transfection efficiency with a relativelty low cytotoxicity, and allows for better controllability and stability of the trasnfectiion conditions.
authors.affiliation: Second Clinical College, Chongqing Medical University, Chongqing 400016, China. E-mail: 77160520@qq.com.
authors.forename: Ji
authors.initials: J
authors.lastname: Li
authors.forename: Zuojin
authors.initials: Z
authors.lastname: Liu
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D017209:Apoptosis; D049109:Cell Proliferation; D005434:Flow Cytometry; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D007728:Kupffer Cells / Q000187:drug effects*; D016086:Lentivirus; D008055:Lipids / Q000737:chemistry; D009687:Nuclear Proteins / Q000235:genetics*; D000078723:Nuclear Receptor Interacting Protein 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; C000627127:NRIP1 protein, human; D009687:Nuclear Proteins; D000078723:Nuclear Receptor Interacting Protein 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering