血清标本检测miR-29a实时荧光定量PCR方法的建立及临床初步应用  

Study on establishment of a real- time fluorescence quantitative PCR technique for detecting miR- 29a in serum and its clinical primary application

作　　者：陈鑫[1] 陈曦[1] 韩霜[1] 臧素纲[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：《临床和实验医学杂志》2016年第1期16-19,共4页Journal of Clinical and Experimental Medicine

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 Green I实时荧光定量PCR方法检测血清miR-29a,并作临床初步应用。方法用Trizol试剂提取血清总RNA。miR-29a ploy(A)聚合酶加尾逆转录获得c DNA,进行SYBR Green I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-29a的表达水平。结果该方法能定量检测血清miR-29a表达水平,熔解曲线呈单峰,PCR扩增产物特异,在10~3copies/μl至10~6copies/μl范围内有良好的线性关系(r^2=0.991),并且检测重复性好。糖尿病患者血清中miR-29a表达水平明显高于健康体检者(P<0.05)。结论所建立的ploy(A)聚合酶加尾SYBR Green I实时荧光定量PCR方法能敏感、特异地检测血清中miR-29a的表达水平,为下一步临床应用的研究奠定了方法学基础。Objective To establish a method of SYBR Green I real- time FQ- PCR by poly（ A） polymerase tailing for determination of miR- 29 a in serum,and to explore it for primary application. Methods Total RNA was extracted from serum by Trizol reagent. miR- 29 a was reversely transcribed into c DNA by tailing poly（ A） polymerase,and then the c DNA was amplified and detected by using SYBR Green I real- time FQ- PCR. A standard curve of dilution series of C. elegans- miR- 39 mimic was generated,and the expression level of miR- 29 a in serum had been quantitatively detected. Results The expression level of miR- 29 a in serum can quantitatively be detected by this method. The melting curve showed a single peak,the product of PCR was specific,with a good linear relationship in the range of 103 copies / μl to 106 copies / μl（ r2=0. 991）,and the detection was repeatable. The expression level of miR- 29 a in diabetic patients was significantly higher than that of healthy controls（ P〈0. 05）. Conclusion The method of SYBR Green I FQ- PCR by poly（ A） polymerase tailing can sensitively and specifically detect the expression level of miR- 29 a in serum. It lays a methodological foundation for further study on clinical application.

关 键 词：SYBR Green I实时荧光定量PCR miR-29a 糖尿病 

分 类 号：R440[医药卫生—诊断学]