文档介绍：Twist基因RT-PCR检测方法的建立
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作者:刘晓燕,王丽,张志霞,李娟,何涛
【摘要】目的建立Twist基因的逆转录聚合酶链式反应(Reverse tran scription polymerase chain reaction,RT-PCR)条件,为探讨Twist功能学研究奠定方法学基础。方法根据Genebank中的Twist(NM_000474)基因序列设计引物,以GAPDH为内参照基因(NM_002046),用RNA simple Total RNA kit提取培养的肾癌细胞总RNA,按照Quantscript RT kit Quant cDNA第一链合成试剂盒说明书合成cDNA,PCR扩增操作按照TAKARA公司的LA Taq with GC Buffer PCR试剂盒说明书,设置PCR反应体系并优化扩增反应条件,琼脂糖凝胶电泳检测扩增结果。结果采用TAKARA的LA Taq with GC buffer PCR试剂盒并通过PCR扩增条件的优化,成功建立了Twist基因的RT-PCR方法,在肾癌细胞系786-0中扩增出大小为150bp左右清晰的目的条带。结论成功摸索出了具有复杂二级结构、富含GC的Twist基因的RT-PCR扩增方法,并证明在肾癌细胞系786-0中存在Twist基因的特异性表达。
【关键词】肿瘤;Twist基因;RT-PCR
The Setup of RT-PCR for Twist Gene in Tumor Cells
Liu Xiaoyan,Wang Li, Zhang Zhixia, Li Juan, He Tao, Laboratory of Molecular Biology, Luzhou Medical College, Luzhou City, Sichuan Province 646000, P. R. China
Abstract Objective To set up the reverse transcription polymerase chain (RT-PCR) method for detecting Twist expression in case to make further researches on its structure and According to the human Twist gen sequence available in Genbank, a pair of primers was designed and the standardization was calibrated by GAPDH gene as an internal control; total RNA of renal carcinoma cell line 786-0 was extracted by simple total RNA kit and the first chain of cDNA posed in accordance with the instruction of Quantscript RT kit Quant cDN