国家自然科学基金(39700050)和China Medical Board(CMB No.99-698)资助项目.
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hSCF cDNA,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hSCF cDNA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hSCF RNA-CRS.测序表明:该RNA-CRS与hSCF mRNA比较,缺失了从第499位至608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hSCF RNA-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/或调控的检测和研究.
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.
谭文斌,聂怡玲,罗赛群,郭小珊,成光杰,陈汉春,朱定尔.定量RT-PCR中人干细胞因子的RNA-CRS的构建[J].生物化学与生物物理进展,2000,27(3):315-318
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