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

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-7，并作临床初步应用。方法用 Trizol 试剂提取血清总 RNA。miR-7 ploy（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103 copies／uL 至106 copies／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.elegans-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.