SYBR green实时荧光定量PCR检测VEGF mRNA方法的建立

Establishment of VEGF mRNA detection by using SYBR green real-time fluorescent quantitative PCR

目的:建立SYBRgreen实时荧光定量PCR检测VEGFmRNA水平的方法,并初步应用于美容相关疾患。方法:构建内参GAPDH质粒,倍比稀释VEGF和GAPDH标准品,建立PCR标准曲线,同时绘制熔解曲线,后应用SYBRGreen实时荧光定量PCR检测正常皮肤、烧伤瘢痕、恶性黑素瘤细胞株A2058和WM793cDNA的VEGFmRNA水平。结果:VEGF、GAPDH的标准曲线,符合线性要求,相关系数r均为1,熔解曲线峰值单一,产物特异度好,四组样本的SYBRgreen荧光定量PCR结果提示瘢痕组织、A2058、WM793的VEGFmRNA水平明显高于正常皮肤。结论:本研究成功建立了VEGFmRNA水平的SYBRgreen荧光定量PCR检测方法。

Objective To establish a method of SYBR green real-time quantitative PCR for detecting mRNA levels of VEGF, and apply the method to cosmetic diseases preliminarily. Methods The recombinant plasmid of GAPDH was constructed as standard control. Standard curves of VEGF and GAPDH were established by using a series dilution of quantified plasmids and the specificity of PCR products was analysed by their melting curves. VEGF mRNA levels of normal skin, burning scar, A2058 and WM793 melanoma cell lines were measured by SYBR green real-time quantitative PCR. Results Two standard curves of VEGF and GAPDH had a linear feature and their R values were both 1. In addition, the melting curve analysis showed a single peak. The results of four groups showed that the VEGF mRNA levels were higher in burning scar tissue, A2058 cells and WM793 cells than that in normal skin tissue. Conclusion The method of detecting VEGF mRNA levels by SYBR green real-time quantitative PCR was established successfully in this study.