定量RTPCR中人干细胞因子的RNACRS的构建  

Construction of Human Stem Cell Factors RNA-CRS in Quantitative RT-PCR.

作　　者：谭文斌[1] 聂怡玲[1] 罗赛群[1] 郭小珊 成光杰[1] 陈汉春[1] 朱定尔[1] public.cs.hn.cn 

机构地区：[1]湖南医科大学分子生物学研究中心,长沙410078

出　　处：《生物化学与生物物理进展》2000年第3期315-318,共4页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金! (3 970 0 0 5 0 );ChinaMedicalBoard资助项目! (CMBNo 99 6 98)

摘　　要：一种适用于定量RT PCR、用ExonucleaseⅢ部分酶切剔除技术 ,构建人干细胞因子 (hSCF)基因的RNA竞争性参考标准 (RNA CRS)的新方法 :用RT PCR技术 ,从HepG2细胞中扩增出全长人hSCFcDNA ,并克隆入质粒pGEM T获重组的pGEMSCF载体 ,经ExonucleaseⅢ和S1核酸酶适当处理 ,以导致hSCFcDNA中一小片段缺失 ,由此获得重组 pGEMSCF模拟体 (mimic) ,经体外转录得到hSCFRNA CRS .测序表明 :该RNA CRS与hSCFmRNA比较 ,缺失了从第 4 99位至 60 8位共 110个核苷酸 ,但二者RT PCR反应可用同一对扩增引物 ,反应动力学极为相似 .这种hSCFRNA CRS可作为一种较理想的竞争性参考标准 ,适用于定量RT PCR中 ,以对重组hSCF在真核细胞中的表达水平进行准确的定量分析 .此方法亦可推广应用于其他真核基因的表达水平及A novel method was developed to prepare an ExonucleaseⅢ partially digesting RNA as a competitive reference standard (RNA CRS) of human stem cell factor (hSCF) gene in quantitative RT PCR: complete hSCF cDNA was already amplified from HepG2 cells using RT PCR and cloned into pGEM T vector. After the recombinant pGEMSCF was treated with Exonuclease Ⅲ and S1 nuclease at a favorable condition to make a limited deletion in hSCF cDNA, the recombinant pGEMSCF mimic was constructed successfully and transcribed in vitro to obtain the RNA CRS. The hSCF RNA CRS with a 110 bp deletion from base 499 to 608 in hSCF cDNA was identified by DNA sequencing and it is suitable to be used as a reliable RNA CRS for the quantitation of the transcriptional expression level of recombinant hSCF in eukaryotic cells by quantitative RT PCR.

关 键 词：基因表达 定量RT-PCR RNA-CRS hSCF 构建 

分 类 号：Q756[生物学—分子生物学] Q786