Identification of reference microRNAs for quantitative expression analysis in porcine peripheral blood mononuclear cells treated with polyinosinic-polycytidylic acid.

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.