Abstract:

Objective To explore an appropriate reference gene for quantitative identification of miRNA in tuberculous pleural effusion (TBPE) and malignant pleural effusions (MPE), using real-time quantitative PCR (qPCR). Methods A total of 78 patients were enrolled, including 47 patients with tuberculous pleurisy and 31 cases of cancer. Total RNA were extracted from the pleural effusion, and then qPCR based on SYBR Green primer were performed to conclude the concentration and amplification efficiency of the reference genes, including miR-16, miR-20a, miR-192, miR-1268, miR-4281, U6 and Cel-miR-39 in PE. The lower the cycle threshold (Ct) value, the higher expression levels of the genes and the more suitable for relative quantitative analysis. Furthermore, the amplification efficiencies of the qPCR primers ranging from 90% to 110% were suitable for relative quantitative analysis. GeNorm (M value), NormFinder (stable value) and BestKeeper softwares (standard deviation and coefficient of variation) were used to analyze the stability of the reference genes. The reference gene was more stable when M value, stability value or standard deviation and coefficient of variation were smaller. Meanwhile, the difference among repeated detections and the difference among various sample process procedures were also analyzed by paried t test. Results Among 78 PE samples, the expression level of miR-1268 (19.16±4.40), Cel-miR-39 (24.17±0.73) and miR-16 (24.52±1.65) were relatively higher, and the amplification efficiency were 90%, 101% and 110%, respectively. The specific melt curves of the 7 genes represented the higher specificity of the qPCR primers, while there is no significant difference among repeated tests or among various sample process procedures. GeNorm (M value=0.994), NormFinder (stability value=0.674) and BestKeeper (standard deviation=0.73,coefficient of variation=3.02%) analyses totally indicated that Cel-miR-39 was the best stable gene among all the samples. Conclusion Cel-miR-39 can be used as the standard reference gene for miRNA quantitative identification in pleural effusion.

Key words: Polymerase chain reaction, microRNAs, Tuberculosis,pleural, Pleural effusion Biological markers