CAJ | 학술논문

目的：建立ploy（A）聚合酶加尾的SYBR Green Ⅰ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cD‐NA ，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‐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 co py／μL范围内有良好的线性关系（ r2＝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 Green Ⅰ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。
목적：건립ploy（A）취합매가미적SYBR Green Ⅰ실시형광정량취합매련반응（PCR）검측혈청miR‐375，병진행림상초보응용。방법용 Trizol시제제취혈청총RNA。miR‐375ploy（A）취합매가미역전록획득cD‐NA ，진행SYBR Green Ⅰ실시정량PCR확증검측。제작C ．elegans‐miR‐39모의물농도제도희석적표준곡선，절대정량혈청중miR‐375적표체수평。결과해방법능정량검측혈청miR‐375표체수평，용해곡선정단봉，PCR확증산물특이，재103～106 co py／μL범위내유량호적선성관계（ r2＝0．997），병차검측중복성호。당뇨병환자혈청중miR‐375표체수평명현고우건강체검자，차이유통계학의의（ P＜0．05）。결론소건립적 ploy （A ）취합매가미SYBR Green Ⅰ실시형광정량PCR능민감、특이지검측혈청중miR‐375적표체수평，위하일보림상응용연구전정료방법학기출。
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 (r2 =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 .