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Identification of reference microRNAs for quantitative expression analysis in porcine peripheral blood mononuclear
1National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China.
Abstract:
Peripheral blood mononuclear cells (PBMCs) are clinically important cells. Detection of microRNAs (miRNAs) expression in PBMCs can be useful for miRNA biomarker discovery for various diseases. Quantitative real-time PCR (qRT-PCR) has become an important method used for measuring miRNAs expression. However, the reliability of qRT-PCR data critically depends on proper selection of reference genes. Here, we performed qRT-PCR to quantify the expression levels of nine miRNAs (Ssc-miR-16, Hsa-miR-25, Ssc-miR-34a, Hsa-miR-93, Bta-miR-92b, Ssc-miR-103, Ssc-miR-106a, Ssc-miR-128 and Ssc-miR-107) and one small nuclear RNA (U6) in PBMCs treated with polyinosinic-polycytidylic acid [poly (I:C)] that widely used for simulating viral infection. We used the four statistical algorithms (GeNorm 3.5, NormFinder, BestKeeper and comparative ∆ Ct method) to evaluate gene expression stability and observed that Ssc-miR-34a was the best single reference gene and the pair of Ssc-miR-107 and Ssc-miR-103 was the best combination of reference miRNAs for porcine PBMCs treated with poly (I:C). Our study shows the first evidence of careful selection of reference miRNAs in porcine PBMCs and maybe helpful for discovering miRNA biomarkers for double-stranded RNA-induced disease.
Insights
Selecting stable reference miRNAs is crucial for accurate gene expression analysis in porcine peripheral blood mononuclear cells (PBMCs). This study identifies Ssc-miR-34a as the best single reference miRNA and Ssc-miR-107/Ssc-miR-103 as the optimal pair for poly (I:C) treated PBMCs.
Area of Science:
- Molecular Biology
- Immunology
- Genomics
Background:
- Peripheral blood mononuclear cells (PBMCs) are vital for disease biomarker discovery.
- MicroRNA (miRNA) expression analysis in PBMCs aids in identifying disease markers.
- Quantitative real-time PCR (qRT-PCR) is a key method for miRNA expression measurement, requiring reliable reference genes.
Purpose of the Study:
- To identify stable reference miRNAs for accurate gene expression analysis in porcine PBMCs stimulated with polyinosinic-polycytidylic acid (poly(I:C)).
- To validate reference miRNA candidates using multiple statistical algorithms for robust selection.
- To support miRNA biomarker discovery for double-stranded RNA-induced diseases.
Main Methods:
- Performed qRT-PCR to quantify expression of nine miRNAs and one small nuclear RNA (U6) in porcine PBMCs.
- Treated PBMCs with poly(I:C) to simulate viral infection.
- Utilized GeNorm, NormFinder, BestKeeper, and comparative ΔCt methods to assess miRNA expression stability.
Main Results:
- Ssc-miR-34a demonstrated the highest expression stability as a single reference gene.
- The combination of Ssc-miR-107 and Ssc-miR-103 was identified as the most stable reference miRNA pair.
- These findings provide crucial validation for reference miRNA selection in porcine PBMCs under specific experimental conditions.
Conclusions:
- Ssc-miR-34a and the pair Ssc-miR-107/Ssc-miR-103 are recommended as reliable reference miRNAs for porcine PBMCs treated with poly(I:C).
- This study establishes a foundation for accurate miRNA profiling in porcine models of viral infection and related diseases.
- The validated reference miRNAs will enhance the reliability of miRNA biomarker discovery in swine immunology and disease research.
