摘要
目的 建立检测RASSF1A(RASassociationdomainfamily 1ARASSF1A)mRNA的SYBRGreenⅠ实时定量PCR的方法 ,了解脑胶质瘤中RASSF1A的表达水平 ,研究RASSF1AmRNA的表达和肿瘤恶性程度的关系。方法 构建克隆载体作为定量模板 ,RocheLightCycler 定量检测 4 8例不同恶性程度胶质瘤组织和 6例正常脑组织标本。结果 4 8例脑胶质瘤组织中有 5例未检出RASSF1AmRNA。与内参照三磷酸甘油醛脱氢酶基因GAPDH(glyceraldehycle 3 phosphatedehydrogenase,GAPDH)比较后 ,RASSF1AmRNA的表达范围从 0 .0 8～ 1.0 0 /μg ,均值为 (0 .5 0± 0 .0 0 4 ) /μg。 6例正常脑组织标本均有RASSF1AmRNA的表达 ,范围从 0 .70～ 1.80 /μg ,均值为 (1.0 5± 0 .0 8) /μg。各期胶质瘤中RASSF1A的mRNA的表达水平均低于正常脑组织 ,但胶质瘤不同病理级别组之间的差异无显著意义。结论 胶质瘤中RASSF1A的mRNA的表达水平均低于正常脑组织。成功地建立了SYBRGreenⅠ实时定量检测RASSF1A基因表达方法 ,为研究RASSF1A表达水平下调的机制、表达水平和肿瘤的恶性程度、转移。
Purpose To establish a SYBR Green Ⅰ quantitative real-time PCR method for detecting the expression of RASSF1A gene (RAS association domain family 1A RASSF1A) in glioma and to investigate the relationship between RASSF1A mRNA expression and tumor grades. Methods Plasmid was cloned for the standard template.SYBR Green Ⅰ quantitative real-time PCR was set up to examine the expression of RASSF1A mRNA in 48 glioma tissues and 6 normal brain tissues using Roche LightCycler . Results Data was normalized by dividing the copy number of the target cDNA by the copy number of GAPDH (glyceraldehycle-3-phosphate dehydrogenase,GAPDH).RASSF1A expression could not be detected in five glioma tissues,other 43 glioma tissues RASSF1A expression levels ranges from 0.08-1.00/μg,mean value was (0.50±0.004)/μg.Six normal brain tissues all showed RASSF1A expression with the range from 0.70-1.80/μg,mean value was (1.05±0.08)/μg.RASSF1A expression levels were lower in different grades glioma than in normal brain tissues,but the expression level was not correlated with the pathologic grade of malignancy. Conclusions The expression of RASSF1A mRNA was lower in glioma than in normal brain tissues.RASSF1A mRNA was successfully detected by SYBR Green Ⅰ quantitative real-time PCR.This method may be used for further study of RASSF1A mRNA low expression mechanism,low expression with tumor grades,malignancy and prognosis in glioma.
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
2004年第6期575-578,共4页
Fudan University Journal of Medical Sciences
