乳腺癌细胞(MCF-7)内参基因的选择

Selection of suitable reference genes for MCF-7 cell line

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.