Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression / 中华创伤杂志(英文版)
Chinese Journal of Traumatology
;
(6): 174-178, 2003.
Article
in English
| WPRIM
| ID: wpr-270338
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Ribonucleases
/
Biological Assay
/
DNA
/
Membrane Glycoproteins
/
Molecular Sequence Data
/
Base Sequence
/
Monocytes
/
Receptors, Immunologic
/
RNA Probes
/
Cells, Cultured
Type of study:
Diagnostic study
Limits:
Humans
Language:
English
Journal:
Chinese Journal of Traumatology
Year:
2003
Type:
Article
Similar
MEDLINE
...
LILACS
LIS