应用cDNA RDA联合cDNA阵列点杂交筛查差异表达基因的方法学分析  

Analysis of isolating differentially expressed genes through cDNA RDA combined with cDNA arrays

作　　者：彭依群[1] 邓小戈[1] 翟木绪[1] 隋国良[1] 王敏[1] 廖二元[1] 

机构地区：[1]中南大学湘雅二医院代谢内分泌研究所,长沙410011

出　　处：《中国骨质疏松杂志》2005年第4期413-417,共5页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金资助(39970374);国家"九五"攻关基金资助(969060505)。

摘　　要：目的探讨采用cDNA代表性差异分析法(representational difference analysis,RDA)联合cDNA阵列点杂交筛查差异表达基因的可行性及局限性。方法将17β雌二醇(E2)干预MG63细胞cDNA分别制备成检测和驱赶扩增子进行消减杂交作为整个cDNARDA的内部质控,以干预组扩增子、对照组扩增子和第4轮双向消减产物为模板,行内对照基因GAPDH的RTPCR进行消减效率分析,Southern杂交证实消减产物来源,快速Northern杂交证实消减产物确实呈差异表达;克隆到pGEMTeasy载体,转化JM109感受态细菌并铺Amp+XgalIPTG琼脂皿得到差异表达文库,随机挑取白色单菌落培养后点成尼龙阵列膜,分别与干预组和对照组扩增子及第4轮消减产物杂交,得到差异表达克隆用于后续研究。结果内部质控结果显示,第3轮消减杂交后,PCR未见差异性产物扩增;cDNARDA共得到3个雌二醇诱导表达上调条带和2个表达下调条带。Southern杂交结果显示在检测扩增子及第1～4轮差异产物中均可见阳性杂交信号,而驱赶扩增子中无杂交信号;快速Northern杂交证实干预组细胞总RNA的两处杂交信号均较对照组强;阵列膜同一克隆两个点对应的杂交信号呈高度相关一致性,共得到120个差异表达克隆(56个表达上调,64个表达下调)。结论cDNA代表性差异分析法联合cDNA阵列点杂交方法能有效快速高通量筛查E2诱导MG63差异表达基因。Objective To investigate the feasibility and limitation of isolating differentially expressed eDNA fragments from human osteoblast-like MG-63 cells induced by 17 beta-estradiol （E2） through modified cDNA representational difference analysis（RDA） combined with eDNA arrays. Methods The internal quality control of RDA was performed by subtractive hybridization of “tester” and “driver” amplicon prepared by cDNA all from E2 treated MG-63 cells. RT-PCR for GAPDH was performed to evaluate the subtractive efficiency of RDA. The differentially expressed cDNA fragments were confirmed by Southern blot and “shotgun” Northern blot. After confirmation,the eDNA fragments were cloned into the pGEM-T easy vector and the subtractive eDNA libraries prepared in E. coli JMI09 cells. The eDNA libraries were grown on LB/Amp^＋/X-gal/IPTG plates and white colonies were picked up randomly and individually grown in LB/amp^＋ medium in 96-well plates. After PCR, colonies were individually blotted onto a Hybond N membrane. The prepared array membranes were hybridized with “tester ” amplicon, “driver” amplicon and the fourth subtractive products to evaluate its usage for further study. Results After the third subtractive hybridization, no amplified products was found in PCR of the internal quality control of RDA. Through eDNA RDA, 3 upregnlated and 2 downregulated fragments were expressed in the fourth subtraction hybridization using eDNA from MG-63 cells induced with and without E2 as “tester” amplicons, respectively. Southern blotting showed that strong hybridization signals were found in “tester” amplicon and the subtractive products, but not in “driver” amplicon .Two hybridization signals from MG-63 cells treated with E2 were stronger than those from MG-63 cells without E2 in “Shotgun” Northem blotting. The two hybridization signals from the same clone showed high consistency on hybridized membranes. Through array dot blotting, we obtained 120 differentially expressed clones including 56 upre

关 键 词：cDNA代表性差异分析法 CDNA阵列 基因表达 方法学 

分 类 号：Q3[生物学—遗传学]