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Measurement of thrombomodulin mRNA expression in brain capillaries by polymerase chain reaction
N D Tran1, L Wang, S S Schreiber
1Department of Neurology, University of Southern California School of Medicine, Los Angeles 90033, USA.
Abstract:
Thrombomodulin (TM), an endothelial integral membrane protein, is a potent activator of the protein C anticoagulant pathway. TM protein expression is limited and regionally distributed in the brain. Recent investigations have demonstrated low TM mRNA expression by brain endothelium, corresponding to its distribution at the protein level. To facilitate the study of TM expression at the transcriptional level, we measured TM mRNA by quantitative-competitive polymerase chain reaction (QC-PCR) and by standard densitometric analysis of reverse transcriptase-PCR products (RT-PCR) in different regions of bovine brain. QC-PCR demonstrated differential TM mRNA expression in the pons (100+/-9%), cerebellum (359+/-103%), and cortex (441+/-24%). We compared these results with those of RT-PCR and found similar differences in relative TM mRNA expression in the pons (100+/-44%), cerebellum (343+/-8%), and cortex (404+/-62%). Data derived by QC-PCR and RT-PCR were highly correlated (r=0.99, p<0.03). These findings indicate that either QC-PCR or RT-PCR can be used to accurately quantify TM mRNA.
Insights
This study quantifies thrombomodulin (TM) mRNA in bovine brain regions using two PCR methods. Both methods revealed significant differences in TM mRNA expression across the pons, cerebellum, and cortex, validating their accuracy.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Thrombomodulin (TM) is an endothelial protein crucial for the protein C anticoagulant pathway.
- TM protein expression in the brain is known to be limited and regionally specific.
- Previous studies indicated low TM mRNA expression in brain endothelium, correlating with protein distribution.
Purpose of the Study:
- To quantify thrombomodulin (TM) mRNA levels in different regions of the bovine brain.
- To compare the efficacy of quantitative-competitive polymerase chain reaction (QC-PCR) and standard reverse transcriptase-PCR (RT-PCR) for TM mRNA measurement.
- To investigate the transcriptional-level expression of TM in the brain.
Main Methods:
- Quantitative-competitive polymerase chain reaction (QC-PCR) was employed to measure TM mRNA.
- Standard densitometric analysis of reverse transcriptase-PCR (RT-PCR) products was used for comparison.
- Bovine brain tissue from different regions (pons, cerebellum, cortex) was analyzed.
Main Results:
- QC-PCR demonstrated differential TM mRNA expression: pons (100+/-9%), cerebellum (359+/-103%), and cortex (441+/-24%).
- RT-PCR showed similar relative TM mRNA expression patterns: pons (100+/-44%), cerebellum (343+/-8%), and cortex (404+/-62%).
- Data from both QC-PCR and RT-PCR were highly correlated (r=0.99, p<0.03).
Conclusions:
- Both QC-PCR and RT-PCR are reliable methods for accurately quantifying thrombomodulin mRNA.
- Significant regional differences in TM mRNA expression exist within the bovine brain.
- These findings facilitate further research into the transcriptional regulation of TM in the brain.
