poly(A)加尾的实时荧光定量PCR检测血清miR-124a表达及临床初步应用

SYBR Green I real-time FQ-PCR by poly(A)polymerase tailing for detecting expression of miR-124a in serum and its clinical primary application

目的建立ploy（A）聚合酶加尾的SYBR Green I实时荧光定量PCR方法检测血清微小RNA（miR）-124a,并作临床初步应用。方法用Trizol试剂提取血清总RNA。miR-124aploy（A）聚合酶加尾逆转录获得cDNA,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,定量血清中miR-124a的表达水平。结果该方法能定量检测血清miR-124a表达水平,熔解曲线呈单峰,PCR扩增产物特异,在103∽106 copies/uL范围内有良好的线性关系（r2=0.999）,并且检测重复性好。糖尿病患者血清中miR-124a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-124a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 fluorescence quantitative PCR（FQ PCR）by poly（A）pol ymerase tailing for the determination of microRNA-124a（miR 124a） in serum,and to preliminarily apply it in clinic. Methods Total RNA was extracted from serum by Trizol reagent, miR-124a was performed the reverse transcription into cDNA by tailing poly（A） polymerase,and then the cDNA was amplified and detected by using SYBR Green I real-time FQ-PCR. The standard curve of a gra- dient dilution series of C. elegans-miR-39 mimic was prepared,and the expression level of miR-124a in serum was quantitatively de- tected. Results The method could quantitatively detect the expression level of miR-124a in serum. The melting curve showed a sin- gle peak, the PCR amplification products were specific, the good linear relationship existed in the range of 103 --106 copies/uL （r2 ： 0. 999） ,and the detection had good repeatability. The expression level of miR-124a in diabetic patients was significantly higher than that in healthy subjects （P〈0.05）. Conclusion The established method of SYBR Green I FQ-PCR by poly（A） polymerase tailing could detect the expression level of serum miR 124a sensitively and specifically,which lays a methodological foundation for further study on its clinical application.