Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression.
- Author:
Yong-hua CHEN
1
;
Jian-xin JIANG
;
Chang-lin LI
;
Dao-jie ZHANG
;
Jian-qiong XIONG
;
Zong-liang ZHANG
;
Pei-fang ZHU
;
Zheng-guo WANG
Author Information
- Publication Type:Journal Article
- MeSH: Antigens, Surface; analysis; Base Sequence; Biological Assay; Cells, Cultured; DNA; analysis; genetics; Gene Expression Profiling; methods; Humans; Lipopolysaccharide Receptors; analysis; Lymphocyte Antigen 96; Membrane Glycoproteins; analysis; Molecular Probe Techniques; Molecular Sequence Data; Monocytes; metabolism; RNA Probes; analysis; genetics; Receptors, Cell Surface; analysis; Receptors, Immunologic; analysis; Ribonucleases; Toll-Like Receptor 4; Toll-Like Receptors
- From: Chinese Journal of Traumatology 2003;6(3):174-178
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo 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.
METHODSThe 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.
RESULTSThe 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.
CONCLUSIONSThese 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.
