乳腺癌细胞(MCF-7)内参基因的选择  

Selection of suitable reference genes for MCF-7 cell line

作　　者：李光超[1] 武小虎[1] 马友记[2] 张勇[1] 赵兴绪[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]甘肃农业大学动物科技学院,甘肃兰州730070

出　　处：《中国兽医学报》2017年第2期278-281,共4页Chinese Journal of Veterinary Science

基　　金："十二五"农村领域国家科技计划资助项目(2011AA10A210)

摘　　要：将稳定转染了真核表达载体pcDNA V5 HisB-rib-esp的乳腺癌细胞(MCF-7)和正常传代细胞分别于10%和20%胎牛血清(FBS)的DMEM条件下培养,连续传代3次以上。运用实时荧光定量PCR(qRT-PCR)技术检测上述4种细胞样品中5种常用看家基因3磷酸甘油醛脱氢酶(GAPDH)、肌动蛋白(β-actin)RNA聚合酶Ⅱ(RPⅡ)、β2微球蛋白(β2-M)、18S核糖体RNA(18SrRNA)的mRNA稳定表达情况,并使用geNorm程序对其稳定性进行评价确定需要的内参基因数目,以选择转染过程中表达最稳定的内参基因。结果表明,5种看家基因表达稳定度M值的排序为:RPⅡ=18SrRNA>β2-M>β-actin>GAPDH,需要2个内参基因(RPⅡ和18SrRNA)共同校正目的基因的表达。RPⅡ和β2-M分别是不同血清下和转染前后表达最稳定的内参。The pcDNA3. 1 V5 HisB plasmid was constructed and transfected into human breast cancer cell line MCF-7, then cultured with the normal cells containing in DMEM medium 10% and 20% fetal bovine serum level for at least three generations. Quantitative real-timc PCR was used to determine mRNA transcription profiles of 5 most common used housekeeping genes（GAPDH,β- actin,RP Ⅱ ,β2-M, 18S rRNA） in these 4 cells,then geNorm program was used to analysize the stability and to choose the optimal number of reference genes. The results showed that the expres- sive stability of 5 reference genes ranked in the following order：RP Ⅱ = 18S rRNA〉β2-M〉β-actin 〉GAPDH and 2 reference genes（RP Ⅱ and 18S rRNA） were needed to normalize mRNA levels between different samples. Our study indicated that RPⅡ and β2-M genes were the most stable reference gene in different serum levels before and after transfection.

关 键 词：MCF-7细胞 稳定转染 内参基因 qRT—PCR geNorm软件 

分 类 号：Q78[生物学—分子生物学]