Wang H, Wang J, Sun S, Wang Y, Guo J, Ning C, Yang K, Liu J-F
National 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.
Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan, China.
Int J Immunogenet. 2015 Jun;42(3):217-25. doi: 10.1111/iji.12198. Epub 2015 Mar 26.
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.
外周血单个核细胞（PBMCs）是临床上重要的细胞。检测PBMCs中微小RNA（miRNAs）的表达对于发现各种疾病的miRNA生物标志物可能有用。定量实时聚合酶链反应（qRT-PCR）已成为用于测量miRNAs表达的重要方法。然而，qRT-PCR数据的可靠性关键取决于参考基因的正确选择。在此，我们进行qRT-PCR以定量九种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和Ssc-miR-107）和一种小核RNA（U6）在经聚肌苷酸-聚胞苷酸[poly（I:C）]处理的PBMCs中的表达水平，poly（I:C）广泛用于模拟病毒感染。我们使用四种统计算法（GeNorm 3.5、NormFinder、BestKeeper和比较∆Ct法）来评估基因表达稳定性，观察到Ssc-miR-34a是最佳的单一参考基因，而Ssc-miR-107和Ssc-miR-103的组合是经poly（I:C）处理的猪PBMCs的最佳参考miRNAs组合。我们的研究首次证明了在猪PBMCs中仔细选择参考miRNAs的证据，可能有助于发现双链RNA诱导疾病的miRNA生物标志物。
