肝损伤相关循环miRNAs检测方法的建立与初步应用  

Establishment and initial application of circulating miRNAs quantitive technique in detecting liver injury

作　　者：王丹妮[1] 黄世峰[1] 府伟灵[2] 张莉萍[1] 

机构地区：[1]重庆医科大学附属第一医院检验科,重庆400016 [2]第三军医大学第一附属医院检验科,重庆400038

出　　处：《中华医院感染学杂志》2011年第10期1984-1987,共4页Chinese Journal of Nosocomiology

基　　金：国家自然科学基金(30973378;81071621);重庆市自然科学基金(CSTC;2009BB5257;2010BB5390);重庆医科大学附属第一医院院内医学科学基金(YXJJ2009-12)

摘　　要：目的建立可靠的循环mi RNA定量技术,并探讨在检测肝损伤相关mi RNA中的应用价值。方法常规收集正常人血清(浆)标本,mirVanaPARIS试剂盒法抽提血清(浆)小RNA,以mi RNA特异引物引导逆转录,通过Taq Man实时荧光定量PCR对内参U6及mi RNAs进行检测。结果常规收集的临床正常血清标本中U6、mi R-122、mi R-192均能实现特异扩增及定量,相应的平均Ct值约分别为33、27和30,对3份不同留置时间人血清标本定量PCR结果显示,mi R-122、mi R-192和U6的丰度相对稳定;临床肝损伤患者循环mi R-122批内和批间差异分别为1.5%、2.5%,用同法检测胆汁淤积肝损伤模型小鼠循环mi R-122结果显示,循环mi R-122在造模后1 d和3 d增高明显。结论实时定量PCR技术为研究mi RNA提供了较好的技术平台。OBJECTIVE To establish a reliable platform for the quantification of circulating miRNA,and explore its application value in the detection of circulating miRNA during liver injury.METHODS Normal serum（plasma） samples were collected,and small RNA was extracted using the mirVana PARIS kit.U6 and target miRNAs were reverse translated by specific primers and real-time fluorescence quantitative polymerase chain reaction was adopted to quantify U6 and target miRNAs.RESULTS U6,miR-122 and miR-192 were amplified and quantified specifically in RNA preparations isolated from routinely collected clinical serum samples,with corresponding Ct values of 33,27 and 30,respectively.Furthermore,the miRNAs were detected from 3 serum samples maintained at room temperature for different time,and quantitative PCR results indicated that the abundance,of miR-122 and miR-192 were stable.The coefficients of variation（CV） of interrun and intrarun assay were 1.5% and 2.5%,respectively.In practical,the levels of miR-122 were significantly higher in 1d and 3d after bile-duct ligation（BDL） than in the sham-operated controls.CONCLUSION Real-time PCR provides a good platform for miRNA research.

关 键 词：循环miRNA 实时荧光定量PCR 肝损伤 

分 类 号：R446[医药卫生—诊断学]