Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression.
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.
Chen
Yong-hua
YH
Research Institute of Surgery, Third Military Medical University, Chongqing 400042, China.
Jiang
Jian-xin
JX
Li
Chang-lin
CL
Zhang
Dao-jie
DJ
Xiong
Jian-qiong
JQ
Zhang
Zong-liang
ZL
Zhu
Pei-fang
PF
Wang
Zheng-guo
ZG
D000954:Antigens, Surface / Q000032:analysis; D001483:Base Sequence; D001681:Biological Assay; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000032:analysis; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism*; D015347:RNA Probes / Q000032:analysis / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000032:analysis; D011971:Receptors, Immunologic / Q000032:analysis; D012260:Ribonucleases; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D000954:Antigens, Surface; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D015347:RNA Probes; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; C048071:endotoxin receptor; D004247:DNA; D012260:Ribonucleases