【摘要】 目的建立并评估检测糖原合酶激酶3β(GSK3β)及其选择性剪接体(GSK3βΔex8+9)mRNA的实时荧光定量PCR(qRT-PCR)法。方法提取慢性粒细胞白血病(CML)和CML急变期患者外周血或/和骨髓总RNA,分别扩增GSK3β及GSK3βΔex8+9目的片段并构建质粒标准品和标准曲线,建立qRT-PCR法并检测其重复性和特异性。结果成功扩增出GSK3β(133 bp)和GSK3βΔex8+9(219 bp)目的条带,建立了检测两目的基因的qRT-PCR法;相应的标准曲线显示循环阈值与模板浓度线性关系良好,相关系数(r)分别为0.997和0.999;两反应体系的重复性结果显示,批内和批间变异系数(CV)均<2%;特异性结果显示,GSK3β反应体系扩增对照组mRNA仅显示GSK3β条带,GSK3βΔex8+9反应体系扩增对照组mRNA均无GSK3βΔex8+9条带出现。结论建立的qRT-PCR法重复性好,特异性强,可用于GSK3β和GSK3βΔex8+9基因表达的定量检测。更多还原

【Abstract】 Objective To establish and evaluate a real-time fluorescence quantitative PCR( qRT-PCR) method for detecting human glycogen synthase kinase 3β( GSK3β) mRNA and its splice variant GSK3βΔex8 + 9. fragment. Methods The target fragments of GSK3β and GSK3βΔex8 + 9 were amplified from the total RNA extracted from the peripheral blood or bone marrow of the patients with chronic myelocytic leukemia( CML) or with the blast crisis of CML,Then,their plasmid standard preparation and standard curve were constructed to establish a qRT-PCR method,and its reproducibility and specificity were evaluated. Results The GSK3β( 133 bp) and GSK3βΔex8 + 9( 219 bp) fragments were amplified and their corresponding qRT-PCR methods were established successfully. The standard curve showed a fine linear relationship between the cycle threshold( CT) and the template concentration,and the coefficients of correlation( r) were 0. 997 and 0. 999,respectively. Both of qRT-PCR methods had high reproducibility,with the intra-assay and inter-assay coefficients of variation( CV) less than 2%. The specificity analysis demonstrated that only 133 bp of target fragment could be amplified from the patients’ mRNA by the qRT-PCR method for detecting GSK3β but not GSK3βΔex8 + 9. Conclusion The established methods have fine reproducibility and specificity,which can be used to quantitatively detect the mRNA levels of GSK3β and GSK3βΔex8 + 9.更多还原