【摘要】 目的评价相对定量RT-PCR方法中不同内对照基因表达水平的稳定性及其对靶基因预后判断的影响程度。方法以36例食管癌患者为例,收集其手术前(B-1)、手术刚结束时(B0)和术后第三天(B+3)的外周血样品。利用实时定量RT-PCR技术,分别检测内对照基因Beta-actin和GAPDH在不同时间的表达水平,并以CEA mRNA作为靶基因,比较基于这两种内对照得出的靶基因相对定量结果用于患者预后判断的差异;标准化的绝对定量结果被作为"预后判断的参照标准"。结果在三个取样时间点,两种内对照基因表达水平的标准偏差依次分别为Beta-actin mR-NA:1.44,1.56和1.92;GAPDH mRNA:3.16,3.28和4.04;两者的总体变异程度分别为9.9%和17.3%。术后一年的复发结果表明,绝对定量法和基于Beta-actin mRNA的相对定量法在预后判断中能够得到一致的结果,而GAPDH mRNA的相对定量结果与复发之间未显示统计学意义的相关性(P>0.05)。结论相对定量RT-PCR法中内对照基因表达水平的变异程度低于10%时,才能保证内对照的有效性以及靶基因测定结果的可比性。基因表达研究时应对所用的内对照基因表达稳定性进行前期评估。更多还原

【Abstract】 Objective To evaluate the stability of expression levels of different endogenous control genes and its influence on the prognosis judge of target gene.Methods Peripheral blood samples from 36 squamous esophageal cancer patients were collected before surgery(B-1),immediately after surgery(B0) and at the third day post-operatively(B+3),respectively.Gene expression levels of endogenous controls beta-actin and GAPDH at all time points were determined by a real-time quantitative RT-PCR technique.The prognosis value of relative quantitative results of target gene CEA mRNA based on the two control genes was analyzed and compared.Absolute quantitative results with normalization strategy were used as "reference standard for prognosis judge".Results The standard deviations from Beta-actin and GAPDH expression levels at above three time points were 1.44,1.56,1.92 and 3.16,3.28,4.04,respectively.Both total variances were 9.9% and 17.3%,respectively.The follow-up of the 36 patients in one year showed that the consistent prognosis results were obtained for the absolute quantitative assay and the relative quantitative assay based on beta-actin mRNA.But there was not statistically significant relationship between the quantitative result based on GAPDH mRNA and the prognosis relapse(P>0.05).Conclusion In the RT-PCR relative quantitative assay,when the variance degree from the expression levels of an endogenous control gene is lower than 10%,validity of the endogenous control gene and comparability of target gene’s quantitative results could be ensured.In studies of gene expression,the expression stability of the selected endogenous control gene should been assessed in advance.更多还原