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Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients
Published on: June 24, 2019
Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression
Yong-hua Chen1, Jian-xin Jiang, Chang-lin Li
1Research Institute of Surgery, Third Military Medical University, Chongqing 400042, China.
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.
Insights
A new ribonuclease protection assay (RPA) multi-probe set was developed to simultaneously detect MD-2, TLR4, and CD14 mRNA expression in human peripheral blood mononuclear cells (PBMCs). This tool reveals transient gene expression changes following LPS stimulation.
Area of Science:
- Molecular Biology
- Immunology
Background:
- Toll-like receptor 4 (TLR4) signaling is crucial in innate immunity.
- MD-2 and CD14 are essential co-receptors for TLR4 activation by lipopolysaccharides (LPS).
- Understanding the dynamic expression of these genes is vital for studying inflammatory responses.
Purpose of the Study:
- To develop a multi-probe ribonuclease protection assay (RPA) template set.
- To enable simultaneous detection of MD-2, TLR4, and CD14 messenger RNA (mRNA) expression.
- To analyze gene expression patterns in human peripheral blood mononuclear cells (PBMCs).
Main Methods:
- cDNA fragments for MD-2, TLR4, and CD14 were amplified using polymerase chain reaction (PCR).
- Cloned fragments into an expression plasmid with a T7 promoter for anti-sense RNA probe synthesis.
- Extracted total RNA from PBMCs and performed RPA to detect target mRNAs.
Main Results:
- The multi-probe template set was validated by sequencing and successfully used in RPA.
- TLR4, MD-2, and CD14 mRNA levels were quantified in human PBMCs.
- A transient decrease in the expression of these three genes was observed 1-3 hours post-LPS stimulation.
Conclusions:
- A novel RPA multi-probe set was successfully constructed.
- This tool allows for simultaneous quantitative determination of TLR4, CD14, and MD-2 mRNA expression.
- The assay is valuable for studying both constitutive and inducible gene expression patterns.
