Real-time PCR assays for detection of melanogenesis-related genes from Chinese traditional medicine components

Abstract

IntroductionMany Chinese traditional medicines provide good therapeutic effects. To screen their pharmacological activities and to discover new bioactive constituents, a SYBR® Green real-time PCR method was established based on information of the melanogenesis-related genes sequences. MethodsSpecific primers were designed according to the sequences of melanogenesis-related genes TYR, TRP-1, TRP-2 and MITF, to establish and optimize a quantitative real-time PCR reaction system based on treatment with 8-methoxypsoralen. Using this system, the effects of Vernonia anthelmintica (L.) Willd injections, Muniziqi granules, Suziafu tablet, Atirilali tablet, Yiti 'erfeili Aikemuaili Migao and Bairesi Migao on the expression of TYR, TRP-1, TRP-2 and MITF in mouse melanoma B16 cells were assessed. ResultsPrimers were designed to be specific to melanogenesis-related genes, and standard curve was constructed using recombinant plasmids containing gene fragments. Cycle thresholds and the template concentrations showed a good linear relationship, the absorption peak of the dissociation curves was single, correlation coefficients were between 0.983 and 0.999, and amplification efficiencies was between 100.95% and 103.05%. A significant positive correlation was observed between the drug concentration (from 25 to 100 μM) and gene expression after 24 h treatment, although Muniziqi granules and Yiti 'erfeili Aikemuaili Migao had not passed this route. ConclusionReal-time PCR was shown to verify compounds with bioactivities that promote melanin production. It also validated that 8-methoxypsoralen and some traditional medicines increased melanin production by increasing the expression of TYR, TRP-1 and TRP-2 and MITF in B16 cells.