Poly（A）加尾实时荧光定量PCR检测血清微RNA-122的表达及其临床初步应用

Poly（A）tailing-based real-time fluorescence quantitative PCR for detection of serum microRNA-122 expression and its clinical primary application

目的：建立Poly（A）加尾SYBR Green I实时荧光定量PCR法检测血清微RNA-122（miR-122）水平，并初步探讨其临床应用价值。方法选取2013年9月至2014年9月本院就诊的糖尿病患者和健康体检者120例为研究对象，分为糖尿病脂质代谢紊乱组（n=40）、糖尿病非脂质代谢紊乱组（n=40）和对照组（n=40）。提取血清总RNA，miR-122 Poly（A）聚合酶加尾逆转录获得cDNA，进行SYBR Green I实时荧光定量PCR扩增。制作C.elegans-miR-39模拟物浓度梯度稀释的标准曲线定量检测血清miR-122水平，进行3组血清miR-122水平的组间比较。结果该方法可定量检测血清miR-122水平，PCR扩增产物特异性好，熔解曲线呈单峰；检测灵敏度为102拷贝/μl，检测线性范围102-107拷贝/μl。高、中、低浓度样本重复性检测的批内变异系数（CV）分别为2.23%、2.48%和2.75%，其批间CV分别为5.35%、5.88%和5.72%，显示该方法具有较好的重复性和稳定性。该方法检测糖尿病脂质代谢紊乱组、糖尿病非脂质代谢紊乱组和对照组的血清miR-122水平分别为（4.85±3.68）×10^3拷贝/μl、（3.06±1.86）×10^3拷贝/μl和（1.03±0.82）×10^3拷贝/μl，糖尿病脂质代谢紊乱组血清miR-122水平高于糖尿病非脂质代谢紊乱组和对照组（均P〈0.01），糖尿病非脂质代谢紊乱组血清miR-122水平高于对照组（P〈0.05）。结论成功建立Poly（A）加尾SYBR Green I实时荧光定量PCR方法检测血清miR-122水平，该方法的灵敏度高、特异性和重复性均较好。

Objective To establish a method of SYBR Green I real-time fluorescence quantitative PCR（FQ-PCR）by Poly（A）tailing for determination of serum microRNA-122（miR-122）level and to preliminarily explore its clinical value. Methods One hundred and twenty subjects who were treated for diabetes or received health checkup in our hospital between September 2013 and September 2014 were included in this study. The subjects were divided into the dyslipidemia diabetic group （n=40）,non-dyslipidemia diabetic group（n=40）and control group（n=40）. Total RNA was extracted from serum samples. MiR-122 was reversely transcribed into cDNA by Poly（A） polymerase tailing,and then the cDNA was amplified by SYBR Green I real-time FQ-PCR. Standard curve of serially diluted concentrations of C.elegans-miR-39 mimics was generated and used for quantitative measurement of serum miR-122 levels. The serum miR-122 levels were compared among the 3 groups. Results FQ-PCR method can quantitatively measure the serum miR-122 levels. The PCR amplification yielded specific products with a single peak on melting curves. The detection sensitivity was 102 copies/μl,and the linear range of detection was 102 to 107 copies/μl. The coefficients of variation（CV）for repetitive measurements of high-,medium-and low-concentration samples were 2.23%,2.48% and 2.75% within-runs,5.35%,5.88% and 5.72% between-runs,respectively, suggesting good reproducibility and stability by using this method. The serum levels of miR-122 as detected by this method were （4.85 ± 3.68）× 10^3 copies/μl in the dyslipidemia diabetic group,（3.06 ± 1.86）× 10^3 copies/μl in the non-dyslipidemia diabetic group,and（1.03±0.82）×10^3 copies/μl in the control group. The serum levels of miR-122 in dyslipidemia diabetic group were higher than that in non-dyslipidemia diabetic group or in the control group（both P〈0.01）,and it’s higher in non-dyslipidemia diabetic group than that in the control group （P〈0.05）. Conclusion Poly （A） tailing-based SYBR Green I real-time FQ-PCR was successfully established for determining serum miR-122 levels,which was with high sensitivity,good specificity and reproducibility.