实时定量PCR测定人皮肤黑色素瘤细胞G6PD的表达

Assay of G6PD Gene Expression in the Human Skin Melanoma by Real-time PCR

目的建立一种灵敏特异、重复性和稳定性好的检测G6PD表达的方法,以探究G6PD与肿瘤的相关性及其机理.方法用Real-time PCR技术测定了野生型、siRNA干扰型和无关序列对照型人皮肤黑色素瘤A375细胞的G6PD基因表达.绘制出β-actin和G6PD的标准曲线,标定了方法的稳定性和重复性,并以内参照β-actin较对G6PD的量.结果β-actin和G6PD标准曲线的回归系数分别为1.086和0.940;稳定性及重复性验证的Ctβ-actin值变异系数分别为2.46%和1.47%,而CtG6PD值变异系数分别为1.76%和1.87%.以野生型A375细胞作对照,无关序列不干扰A375细胞内源性G6PD基因表达(P>0.05),针对G6PD基因表达的siRNA的干扰效率为49%(P<0.01).结论实时荧光定量PCR在检测细胞G6PD基因表达时具有高灵敏度、高重复性和高稳定性的特点,可进一步用于G6PD与肿瘤的相关性研究.

Objective To explore the relationship between G6PD and cancer and its fundamental principles by establishing a sensitive, duplicable and stable system of detecting G6PD mRNA expression. Methods Real- time PCR was used to detect G6PD mRNA expression in the human skin melanoma （A375） cells with wild type, siRNA interference type and siRNA negative sequences type. The standard curves of β-actin and G6PD were drawn, and the sensitivity, stability and repetition of this method were evaluated, and G6PD mRNA expression was corrected by β- actin gene expression. Results The regression coefficients of the standard curves with β- actin and G6PD were 1. 086 and 0. 940 ; the coefficient variations （CV） of repetition and stability for Ctβ- actin value were 1.76% and 1.87% , and which were 1.76% and 1.87% for CtC6PD ; compared with A375 cells in the wild type, the negative siRNA sequences did not interfere the G6PD mRNA expression （ P〉0.05 ） and the interference efficiency of the siRNA was 49 % （P〈0.01 ）. Conclusions Real - time PCR is Of high sensitivity, reproducibility and stability, and can be used to study the dependability between the G6PD and cancer.