Selection of the most effective small interfering RNA which inhibits the expression of TLR4 in microglial cells and detection of the cytotoxicity of transfection complex
- VernacularTitle:有效抑制小胶质细胞上TLR4表达的siRNA筛选及转染复合物细胞毒性的检测
- Author:
Silan LIU
;
Jianping YANG
;
Lina WANG
;
Lei LIU
;
Caifang LI
;
Chunguang REN
;
Jing ZHOU
;
Wei LI
;
Miao JIANG
;
Zhenni MA
;
Qiaocheng QIU
- Publication Type:Journal Article
- Keywords:
microglial;
Toll-like receptor 4;
small interfering RNA;
select;
transfection complex;
cytotoxicity
- From:
Chinese Pharmacological Bulletin
2010;26(4):457-461
- CountryChina
- Language:Chinese
-
Abstract:
Aim To pick out the siRNA which could most effectively inhibit the expression of TLR4 in microglial cells and to detect the cytotoxicity of the transfection complex.Methods Five siRNAs were chemicaly synthesized:four of them were used to inhibit TLR4 expression in microglial cells,the rest was fluorescence-labeled mismatch siRNA as a nagative control.They were all transfected into microglial cells,respectively.TLR4 mRNA was detected 24 h after transfection by RT-PCR and its protein expression wasobserved by Western blot 48 h later.The cytotoxicity of complex was detected using MTT.Results ① The transfection rate was high enough in microglial cells with siRNA(40 pmol)and LipofectamineTM 2000(1 μl).② The TLR4 siRNA pool reduced TLR4 mRNA by 85%(siRNA_(439)),73%(siRNA_(312)),67%(siRNA_(1495))and 33%(siRNA_(2062))respectively compared with mismatch siRNA-treated group 24 h after transfection in a microglial cell line.③ The TLR4 siRNA439 was the most effective siRNA(P<0.01).④ The cell survival rates were above 85% in the groups of Lipofectamine~(TM) 2000 1 μl compound less than 40 nmol·L~(-1) siRNA.Conclusions ① The TLR4 siRNA_(439) can inhibit TLR4 expression most effectively in microglial.② 40 nmol·L~(-1) siRNA and 1 μl Lipofectamine~(TM) 2000 have low cytotoxicity,which are suitable for transfection.
