实时荧光定量PCR检测血清miR-7方法的建立及临床初步应用

Establishment of a real-time fluorescence quantitative PCR method for detecting miR-7 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miR-7,并作临床初步应用。方法用Trizol试剂提取血清总RNA。miR-7ploy(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,绝对定量血清中miR-7的表达水平。结果该方法能定量检测血清miR-7表达水平,熔解曲线呈单峰,PCR扩增产物特异,在10~3copies/uL至10~6copies/uL范围内有良好的线性关系(r^2=0.995),并且检测重复性好。糖尿病患者血清中miR-7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-7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）polymerase tailing for the determination of miR-7 in serum,and apply it primarily. Methods Total RNA was extracted from serum by Trizol reagent, miR-7 was reverse transcripted into cDNA by tailing poly（A）polymerase, and then the cDNA was amplified and detected by using SYBR Green I real-time FQ-PCR. Generate a standard curve of a dilution series of C. eIegans-miR-39 mimic,and detect the expression level of miR-7 in serum quantitatively. Results The method can quantitatively detect the expression level of miR-7 in serum. The melting curve showed a single peak, the PCR products was specific,a good linear relationship in the range of 103 copies/uL to 106 copies/uL （r^2 = 0. 995）, and the detection was repeatable. The expression level of miR-7 in diabetic patients was significantly higher than those in healthy subjects （P〈0.05）. Conclusion The method of SYBR Green I FQ-PCR by poly（A）polymerase tailing can detect the expression level of miR-7 sensitively and specifically in serum. It lays a methodological foundation for further study on clinical application.