实时荧光定量PCR检测血清miR-375表达及临床应用  

Real-time fluorescence quantitative PCR for detecting expression of serum miR-375 and its clinical application

作　　者：陈鑫[1] 韩霜[1] 臧素纲[1] 宣世海[1] 汪晓莺[2] 周玉贵[1] 

机构地区：[1]南通大学附属东台医院检验科,江苏东台224200 [2]南通大学医学院免疫教研室,江苏南通226001

出　　处：《检验医学与临床》2015年第9期1208-1210,共3页Laboratory Medicine and Clinic

基　　金：江苏省盐城市医学科技发展计划项目(YK20130103)

摘　　要：目的建立ploy（A）聚合酶加尾的SYBR GreenⅠ实时荧光定量聚合酶链反应（PCR）检测血清miR-375,并进行临床初步应用。方法用Trizol试剂提取血清总RNA。miR-375ploy（A）聚合酶加尾逆转录获得cDNA,进行SYBR GreenⅠ实时定量PCR扩增检测。制作C.elegans-miR-39模拟物浓度梯度稀释的标准曲线,绝对定量血清中miR-375的表达水平。结果该方法能定量检测血清miR-375表达水平,熔解曲线呈单峰,PCR扩增产物特异,在103-106 copy/μL范围内有良好的线性关系（r^2=0.997）,并且检测重复性好。糖尿病患者血清中miR-375表达水平明显高于健康体检者,差异有统计学意义（P〈0.05）。结论所建立的ploy（A）聚合酶加尾SYBR GreenⅠ实时荧光定量PCR能敏感、特异地检测血清中miR-375的表达水平,为下一步临床应用研究奠定了方法学基础。Objective To establish a method of SYBR Green I real‐time fluorescence quantitative PCR （FQ‐PCR）by poly（A）polymerase tailing for the determination of miR‐375 in serum and to conduct the preliminary clinical application .Methods Total RNA was extracted from serum by Trizol reagent .miR‐375 was reversely transcripted into cDNA by tailing poly（A）polymerase ,and then the cDNA was amplified and detected by using SYBR Green I re‐al‐time FQ‐PCR .To generate a standard curve of a dilution series of C .elegans‐miR‐39 mimic ,and detect the expres‐sion level of miR‐375 in serum quantitatively .Results The method could quantitatively detect the expression level of miR‐375 in serum .The melting curve showed a single peak ,the PCR amplification products were specific ,a good line‐ar relationship was showed in the range of 103 -106 copies/uL （r^2 =0 .997） ,and the detection had good repeatability . The expression level of miR‐375 in diabetic patients was significantly higher than that in healthy subjects ,the differ‐ence was statistically significant （P〈0 .05） .Conclusion The established method of SYBR Green I FQ‐PCR by poly （A）polymerase tailing can detect the expression level of serum miR‐375 sensitively and specifically and lays a meth‐odological foundation for further study on its clinical application .

关 键 词：SYBR Green I实时荧光定量聚合酶链反应 miR-375 糖尿病 

分 类 号：R446.5[医药卫生—诊断学]