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Updated: Jun 5, 2026
Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease
Published on: July 3, 2018
The quantification of ADAMTS4 and 8 expression and selection of reference genes for quantitative real-time PCR
Chuanyan Zhao1, Yanping Zha, Xiaoyan Wu
1Department of Cardiology, Shandong Provincial Hospital, Shandong University, 324, Jingwu Road, Jinan 250021, PR China.
Introduction:
ADAMTS4 and ADAMTS8 are proteases involved in ECM proteolysis and antiangiogenesis, but little is known about their expression and function in myocardial infarction (MI). We examined ADAMTS4 and ADAMTS8 expression in a rat MI model by quantitative real-time polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). The expressions of glyseraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), acidic ribosomal phosphoprotein P0 (ARBP), and ribosomal protein L13A (RPL13A) were examined in order to validate the appropriate housekeeping genes after MI.
Methods:
Male Wistar rats were subjected to MI, and infarcted myocardial tissue was collected at 3, 6, 12, 24h, 3, 7, 14 and 21days after MI. ADAMTS4, ADAMTS8, and the four housekeeping genes were quantified using qPCR and the expression stability of the four housekeeping genes was investigated using GeNorm software. The protein levels of ADAMTS4 were detected using ELISA kits.
Results:
The M values of GAPDH, ACTB, ARBP and RPL13A were 0.721, 1.2, 0.812 and 0.812 respectively. GAPDH and ARBP were ranked the most stable genes. ADAMTS4 mRNA increased at 3h after MI, peaked at 6h, then decreased rapidly. ADAMTS8 mRNA increased at 6h, peaked at 24h, remained high at 3d, then decreased gradually. The protein levels of ADAMTS4 were significantly increased at 6h, 12h, 24h and 3d after MI.
Conclusion:
The results suggest that GAPDH and ARBP are two appropriate housekeeping genes for the rat MI model. Both ADAMTS4 and ADAMTS8 mRNA levels and ADAMTS4 protein level increased, but they exhibited different expression profiles.
Insights
This study investigated ADAMTS4 and ADAMTS8 expression in rat myocardial infarction (MI) models. Results show distinct mRNA and protein level changes for ADAMTS4 and ADAMTS8 following MI, identifying GAPDH and ARBP as stable housekeeping genes.
Area of Science:
- Cardiovascular Biology
- Molecular Biology
- Biochemistry
Background:
- ADAMTS4 and ADAMTS8 are proteases implicated in extracellular matrix remodeling and angiogenesis.
- Their specific roles and expression patterns in myocardial infarction (MI) remain largely uncharacterized.
- Understanding these proteases is crucial for developing novel therapeutic strategies for heart disease.
Purpose of the Study:
- To investigate the expression profiles of ADAMTS4 and ADAMTS8 in a rat model of myocardial infarction (MI).
- To identify appropriate housekeeping genes for accurate gene expression analysis in MI.
- To elucidate the temporal changes in ADAMTS4 and ADAMTS8 mRNA and protein levels post-MI.
Main Methods:
- Myocardial infarction was induced in male Wistar rats.
- Quantitative real-time polymerase chain reaction (qPCR) was used to measure mRNA levels of ADAMTS4, ADAMTS8, and four candidate housekeeping genes (GAPDH, ACTB, ARBP, RPL13A).
- Protein levels of ADAMTS4 were quantified using enzyme-linked immunosorbent assay (ELISA).
- GeNorm software was utilized to assess the stability of housekeeping genes.
Main Results:
- GAPDH and ARBP were identified as the most stable housekeeping genes for normalization in the rat MI model.
- ADAMTS4 mRNA levels showed a rapid increase, peaking at 6 hours post-MI.
- ADAMTS8 mRNA levels increased more gradually, peaking at 24 hours and remaining elevated for 3 days.
- ADAMTS4 protein levels were significantly elevated at multiple time points after MI, including 6, 12, 24 hours, and 3 days.
Conclusions:
- GAPDH and ARBP are validated as suitable housekeeping genes for qPCR studies in rat MI models.
- Both ADAMTS4 and ADAMTS8 expression is upregulated following MI, but with distinct temporal dynamics at the mRNA level.
- ADAMTS4 protein levels are also significantly increased post-MI, suggesting a role in cardiac tissue remodeling and repair.
