荧光定量RT-PCR检测神经母细胞瘤细胞MYCN基因mRNA的表达  

Detection of MYCN mRNA in neuroblastoma cell lines by quantitative RT-PCR

作　　者：冯晨[1] 唐锁勤[1] 王建文[1] 刘立真[1] 高晓宁[1] 龙卉[1] 

机构地区：[1]中国人民解放军总医院小儿内科,北京100853

出　　处：《中国当代儿科杂志》2007年第1期47-50,共4页Chinese Journal of Contemporary Pediatrics

基　　金：国家自然科学基金(编号30471802)

摘　　要：目的MYCN基因表达对神经母细胞瘤的治疗及预后评估有指导意义,目前国内对于MYCN基因mRNA的定量检测未见报道,该研究拟采用SYBR绿色荧光染料Ⅰ(SYBRGREENⅠ)实时检测的逆转录-聚合酶链反应(RT-PCR)方法,检测神经母细胞瘤细胞系LA-N-5细胞MYCN基因mRNA的表达,并对其可行性及实用性进行研究,力争探索出微量瘤标本的MYCN基因mRNA定量检测的可行方法。方法提取神经母细胞瘤细胞系LA-N-5细胞总RNA,采用SYBRGREENI实时检测的RT-PCR检测其MYCN基因mRNA的表达,并用3-磷酸甘油醛脱氢酶(GAPDH)作为内参照,将MYCN基因的mRNA拷贝数与GAPDH的拷贝数相除,结果为单细胞MYCN基因mRNA的表达水平。结果反应标准曲线有良好的相关性(R2>0.99),PCR产物特异,神经母细胞瘤细胞系LA-N-5MYCN基因mRNA的表达水平为17.4±1.2。结论只要严格控制PCR反应条件,SYBRGREENⅠ定量RT-PCR法可以作为一种良好的定量PCR方法对神经母细胞瘤细胞系LA-N-5MYCN基因mRNA的表达进行检测,此方法为临床微量神经母细胞瘤瘤组织的MYCN基因mRNA的定量检测提供了可能。Objective To examine the feasibility and practicability of quantitative reverse transcriptase-polymerase chain reaction （RT-PCR） with SYBR GREEN I fluorescence for detecting the MYCN mRNA expression in neuroblastoma cell line LA-N-5. Methods MYCN mRNA expression in LA-N-5 cells was measured using real time RT-PCR with SYBR GREEN I. Glyceraldehyde phosphate dehydrogenase （ GAPDH ） was used as internal control. The level of the MYCN mRNA was calculated as MYCN copies/GAPDH copies. Results Standard curves were linear and showed high correlations （ R2 〉 0.99 ）. The ratio of MYCN mRNA copies to GAPDH mRNA copies was calculated based on specific PCR products. The MYCN mRNA level in LA-N-5 cells was obtained （ 17.4 ± 1.2 ）. Conclusions Quantitative RT-PCR with SYBR GREEN I fluorescence may be a sensitive and reliable method for detecting the MYCN mRNA expression. It may also be potential applicable for detecting the MYCN mRNA expression in the small amount neuroblastoma tissues.

关 键 词：神经母细胞瘤 MYCN 荧光定量RT—PCR 绿色荧光染料Ⅰ 

分 类 号：R739.4[医药卫生—肿瘤]