Reproducibility of quantitative real-time PCR assay in microRNA expression profiling and comparison with microarray analysis in narcolepsy

Zhenhua Liu, Liling Yang, Yingzi Zhao, Minglu Tang, Fumin Wang, Xiaoting Wang, Guanzhen Li, Yifeng Du

MicroRNAs (miRNAs) have been shown in the pathogenesis of human neurological disorders. The study aims to identify the involvement of miRNAs in the pathophysiology of narcolepsy. Here, we conducted three independent high-throughput analysis of miRNA (miRNA microarray) in peripheral blood from 20 narcolepsy patients who fulfilled the criteria compared to 20 healthy controls with validation experiment using quantitative real-time polymerase chain reaction (real-time PCR) panels. By analyzing 2805 miRNAs in peripheral blood with microarray we identified 128 miRNAs (105 high expression and 23 low expression) that were different in patients with narcolepsy in comparison with healthy control. Then we chose six high expression candidates and six low expression candidates of at least twofold difference and p value < 0.05 to validate the changes in three independent experiments in vitro using real-time PCR. The validation test showed that levels of hsa-mir-1267, hsa-miR-4309, hsa-miR-554, hsa-miR-1272, hsa-miR-4501, hsa-miR-182-3p were higher, whereas the level of hsa-miR-625-5p, hsa-miR-100-5p, hsa-miR-125b-5p, hsa-miR-197-3p, hsa-miR-4522, hsa-miR-493-5p was lower in narcolepsy patients than healthy controls. The levels of 12 miRNAs differed significantly in peripheral blood from narcolepsy patients which suggested that alterations of miRNAs expression may be involved in the pathophysiology of narcolepsy.