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

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.