摘要
目的比较人不同孕期胎盘组织中最常用的内参基因及其他几种管家基因mRNA表达水平的差异及稳定性,选出用于该研究的最佳内参基因。方法应用荧光实时定量RT-PCR技术检测6种管家基因[3磷酸甘油醛脱氢酶(GAPDH)、β肌动蛋白(β-actin)、β2微球蛋白(β2-M)、18S核糖体RNA(18srRNA)、RNA聚合酶Ⅱ(RPⅡ)、泛素(UBC)]在早、中、晚不同孕期孕妇绒毛和胎盘组织中mRNA的表达情况,并应用geNorm程序对内参基因表达稳定性进行分析。结果在早、中、晚不同孕期的孕妇早孕绒毛或胎盘组织中内参基因GAPDH mRNA的表达水平变化最大,其中在早孕绒毛组织中表达水平最高,随着孕期的延长其表达水平逐渐降低;内参基因β-actin mRNA转录水平则呈相反的趋势变化,标本间mRNA表达水平变化亦较大;内参基因β2-M、18S和UBC mRNA表达水平在不同孕期标本间变化相对较小,其中内参基因RPⅡmRNA表达水平在不同孕期标本间变化最小。所选内参基因的表达稳定度由高到低依次为RPⅡ>UBC>18srRNA>β2-M>β-actin>GAPDH。结论在应用荧光实时定量RT-PCR技术研究基因转录表达时,应根据研究所用的材料选择合适的内参基因;本研究中RPⅡ是可用的稳定内参基因。
Objective To compare differences in the mRNA expression levels of the most commonly used reference genes and housekeeping genes and stability of villi and placenta tissue during early, middle and late pregnancy, and to select the optimal reference gene for the study. Methods Quantitative real-time PCR was used to determine mRNA transcription profiles of 6 housekeeping genes （GAPDH, β-actin, RPⅡ, 18SrRNA, β2-M, and UBC） of early pregnant villi and placenta tissue during early, middle and late pregnancy. Then we compared their transcription stabilities by GeNorm analysis program and choose the optimal reference gene. Results The mRNA expression of reference gene GAPDH of early pregnant villi and placenta tissue changed most significantly during each pregnancy; it was the highest in early pregnancy villus and gradually decreased with the extended duration of pregnancy. The expression of β-actin mRNA showed the opposite trend and changed significantly. Reference genes β2-M, 18s rRNA and UBC mRNA expressions changed relatively less between different samples. The expression of RP Ⅱ was the most stable. The stability of these genes ranked in the following order： RP Ⅱ〉UBC 〉18s rRNA〉β2- M〉β-actin〉GAPDH. Oonclusion Choosing an appropriate reference gene is a prerequisite for accurate RT-PCR expression profiling. Our study indicates that RNA polymerase II gene is a stable reference gene that can be used.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2014年第2期218-221,共4页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.30800909)~~
