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目的建立血浆循环MicroRNA的提取检测方法,并且检测其在冠心病患者和健康人群之间的差异表达,寻找新的疾病诊断标记物。方法前期入选收取15例冠心病患者和15例健康人的血样标本,性别和年龄在统计学上没有差异,采用Trizol LS提取血浆中的总RNA,Nan-oDrop 1000检测总RNA浓度,设计特异性引物检测hsamiR-16、hsa-miR-126的表达,采用外加cel-miR-39进行校正,分析血浆中循环miRNA的表达差异。结果 (1)血浆提取的总RNA在280nm处有最大吸收峰,260 nm/280 nm比值介于1.67-2.17之间;(2)检测方法的特异性和灵敏度较好,cDNA梯度稀释20倍可以分辨出来,用hsa-miR-16 mimics的检测累加特异性,其CT值成梯度的累加,特异性较好;(3)检测hsa-miR-126在冠心病患者和健康人的表达有统计学差异(P<0.001),hsa-miR-16在两组间的表达没有统计学差异(P=0.306)。结论采用Trizol LS提取血浆总RNA的方法可行,茎环实时定量荧光检测miRNAs的方法特异性和灵敏性较好,血浆hsa-miR-126表达的差异可能在冠心病的发展过程中发挥调节作用,其具体的机制还有待于进一步的研究。
OBJECTIVE: To establish a method for the extraction and detection of plasma circulating MicroRNAs and to detect the differential expression of microRNAs in patients with coronary heart disease and healthy individuals, in search of new diagnostic markers for diseases. Methods Blood samples were collected from 15 patients with coronary heart disease and 15 healthy subjects in the early stage. There was no statistical difference in sex and age. Trizol LS was used to extract total RNA from plasma. Nan-oDrop 1000 was used to detect the total RNA concentration and the specificity Primers were used to detect hsamiR-16 and hsa-miR-126 expression, and cel-miR-39 was used to correct the expression of circulating miRNA in plasma. Results (1) The total RNA extracted from plasma had the maximum absorption at 280 nm and the ratio of 260 nm / 280 nm was between 1.67 and 2.17. (2) The specificity and sensitivity of the detection method were better. The specificity of hsa-miR-16 mimics detection was additive, and its CT value was gradient plus. The difference was statistically significant. (3) The expression of hsa-miR-126 in patients with coronary heart disease and healthy people was statistically significant (P <0.001). There was no significant difference in hsa-miR-16 expression between the two groups (P = 0.306). Conclusion The method of Trizol LS extraction of total RNA in plasma is feasible. The specificity and sensitivity of stem-loop real-time quantitative fluorescence detection of miRNAs are better. The difference of plasma hsa-miR-126 expression may play a regulatory role in the development of coronary heart disease, The specific mechanism remains to be further studied.