| Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression |
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| 作者姓名： | 陈永华 蒋建新 李昌麟 张道杰 熊建琼 张宗梁 朱佩芳 王正国 |
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| 作者单位： | [1]ResearchInstituteofSurgery,ThirdMilitaryMedicalUniversity,Chongqing400042,China [2]ShanghaiInstituteofBiochemistryandCellBiology,ChineseAcademyofSciences,Shanghai200031,China |
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| 基金项目： | SupportedbytheNational“ 973Project” (No.G1 9990 542 0 3) |
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| 摘 要： | Objective: To construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods : The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR)using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7/ promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method. Results: The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation. Conclusions: These new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.
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| 关 键 词： | 复合探针核糖核酸酶保护试验 内毒素 受体 基因表达 外周血 PCR RNA |
Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression |
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| CHEN Yong hua,JIANG Jian xin ,LI Chang lin,ZHANG Dao jie,XIONG Jian qiong,ZHANG Zong liang,ZHU Pei fang and WANG Zheng guo.Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression[J].Chinese Journal of Traumatology(English Edition),2003,6(3):174-178. |
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| Authors: | CHEN Yong hua JIANG Jian xin LI Chang lin ZHANG Dao jie XIONG Jian qiong ZHANG Zong liang ZHU Pei fang and WANG Zheng guo |
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| Institution: | 1. Research Institute of Surgery, Third Military Medical University, Chongqing 400042, China
2. Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China |
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| Abstract: | Objective: To construct the multi probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD 2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods: The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method. Results: The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD 2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1 3 hours after 100 ng/ml LPS stimulation. Conclusions: These new RPA multi probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD 2 mRNAs in both constitutive and inducible types. |
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| Keywords: | Lipopolysaccharide Receptor Gene expression CD14 |
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