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

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(SYBR GREEN Ⅰ) 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SYBR GREEN I 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-5 MYCN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，SYBR GREEN Ⅰ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-5 MYCN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。［中国当代儿科杂志，2007，9（1）：47-50］

Abstract:

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.［Chin J Contemp Pediatr, 2007, 9 (1):47-50］