半定量RT-PCR检测胶质瘤细胞分化相关新基因的表达

Analysis of Novel Differentiation Related Genes in Gliomas by RT-PCR

背景与目的:利用基因芯片和生物信息学手段从大量的基因中筛选肿瘤发生发展过程中重要的调控基因,是目前功能基因组研究的一个重要研究方向。但是小样本量的检测有时会出现一些假阳性和假阴性结果,因此很有必要抽取部分样本的基因进行验证。本文利用半定量Rt-PCR检测了从胶质瘤恶性进展和诱导分化两个基因表达谱中利用生物芯片和生物信息学技术筛选出来的7条基因在胶质瘤组织中的表达情况,目的在于,一方面验证以前的基因芯片数据的可靠性;另一方面通过对基因表达水平的半定量,进行横向的对比,希望能够找出这些基因在不同级别胶质瘤中的表达规律,为进一步的功能研究提供依据。方法:按照7条基因的Gen-Bank登录号,检索GenBank得到这些基因的全长cDNA序列,然后设计相应的PCR引物,针对22例恶性胶质瘤病人的肿瘤组织作检测,22例胶质瘤按照WHO分级其中Ⅰ级3例,Ⅱ级8例,Ⅲ级7例,Ⅳ级4例,最后利用SmartView电泳图像分析软件对各个基因在不同组织中的表达进行半定量。结果:这些基因在这些肿瘤中的表达差异是有规律性的,和以前的基因芯片结果基本一致。如X54304(肌球蛋白轻链)、A1264216(胆碱转运因子2)都是随着恶性程度的进展而表达升高,而NM_019896(DNA多聚酶p12亚基)、NM_015962(CGI-35蛋白)、AB037886(NESH蛋白)、D38305(Tob蛋白)、M87507(半胱天冬酶1,Caspasel)都是随着恶性程度的进展而表达降低。结论:我们对于基因芯片和生物信息学技术筛选到的7条基因作半定量RT-PCR分析,证实我们使用的基因芯片是可靠的;同时显示这些基因在胶质瘤的恶性进展过程中都发生了规律性的改变,信息学检索提示其中有些基因可能是肿瘤恶变的重要调控因子。

BACKGROUND & OBJECTIVES: Exploring the transcriptional regulation mechanism by microar-ray and bioinformatic analysis is an important technology in genomic research field.However, those methods need to be validated because they have high false positive and false negative rate.In order to discover tumor development related genes, we used RT-PCR to analyze 7 novel differentiation related genes expression in glioma tissues of different grades.METHODS: Firstly, PCR primers were designed by Primer3 software following retrieval of those gene sequences in Gen- Bank.Subsequently, 22 glioma tissue samples of different grades (WHOⅠ, 3; WHOⅡ, 8; WHOⅢ, 7; WHOⅣ, 4.) were analyzed by RT-PCR.Finally, the genes expression levels ware quantified by SmartView Image processing software, and analyzed.RESULTS: The expression profiles of those genes were coincident with the results that we have obtained by microarray analysis.X54304(myosin, light polypeptide) and AI264216(CTL2 gene) were up-regulated with raising tumor grade.On the contrary, NM_019896(DNA polymerase epsilon pl2 subunit), NM_015962(CGI-35 protein), AB037886(NESH protein), D38305(Tob protein), and M87507(Caspasel) were down-regulated with increase in tumor grade.CONCLUSION: The expression profile of those 7 genes determined by RT-PCR is similar to the results of microarray and bioinformatic analysis.It suggests that the microarray technique which we previously applied is reliable.It also indicated that there may be a gene regulation network, within which some genes may be important regulators in the development of gliomas.