小鼠CCL20 mRNA实时荧光定量PCR检测方法的建立

目的:建立检测小鼠脂肪组织中趋化因子CCL20(CC chemokine ligand 20)mRNA的实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)的方法。方法:分离小鼠脂肪组织细胞,经PMA和离子霉素刺激4 h后收集细胞提取总RNA并逆转录为cDNA。采用qRT-PCR方法检测趋化因子CCL20 mRNA的表达水平。评价该方法的特异性,同时应用该方法检测小鼠脂肪组织中趋化因子CCL20mRNA相对表达水平。结果:建立了小鼠脂肪组织中趋化因子CCL20的SYBR Green实时荧光定量PCR检测方法,结果显示该方法的熔解曲线为单峰,同时核酸电泳显示一条特异性条带。检测结果表明高脂饮食诱导的肥胖小鼠脂肪组织中趋化因子CCL20的相对表达水平明显高于正常饮食对照组(t=3.02,P<0.05)。结论:建立了检测小鼠脂肪组织中趋化因子CCL20 mRNA的实时荧光定量PCR方法,特异性好、灵敏性强,为进一步研究小鼠CCL20的生物学功能提供了实验基础。

Establishment of the real-time fluorescence quantitative PCR assay for detection of murine adipose tissue chemokine CCL20 mRNA expression

Objective: To establish SYBR Green Supermix based real-time fluorescence quantitative PCR(qRT-PCR)method for detection of chemokine CCL20 mRNA expression.Methods: The total cells of murine adipose tissue were separated,and then incubated them with PMA and ionomycin at 37℃ for 4 h,total RNAs were extracted from the separated cells and then transcribed reversely into cDNAs.The relative expression level of mouse chemokine CCL20 mRNA in adipose tissue was detected by qRT-PCR.The sensitivity and specificity of this method were evaluated.Results: The melting curve showed single peak and the amplified products were electrophoresed and also detected a single product.Compared to ND mice,the CCL20 mRNA relative expression was significantly increased in HFD mice adipose tissue(t=3.02,P<0.05).Conclusion: The method to detect mouse chemokine CCL20 mRNA in adipose tissue by SYBR Green based qRT-PCR was good in specificity and sensitivity.It can be used as a standard method of qRT-PCR for detection of mouse chemokine CCL20 expression.