茎环引物fqRT-PCR检测心肌梗死后重构患者血清miRNA-21的表达及其临床意义

Stem-loop primers based fqRT-PCR detection for serum miRNA-21 expression of patients suffering myocardial remodeling after infarction and its clinical significance

目的建立茎环引物实时荧光定量逆转录-聚合酶链反应(fqRT-PCR)检测微小RNA(miRNA)的定量检测方法。方法选择300例心肌梗死患者作为心肌梗死组,心肌梗死组患者根据病情再分为急性组、亚急性组及慢性组;同期选取100例健康体检者作为对照组。采用茎环引物fqRT-PCR、金纳米-分子信标fqRT-PCR检测其血清miRNA-21表达水平。结果急性期、慢性期心肌梗死患者血清miRNA-21表达水平高于亚急性期和对照组(P<0.05),亚急性期患者的血清miRNA-21表达水平高于对照组(P<0.05)。茎环引物fqRT-PCR检测miRNA-21,最低检出102 copies/μL的稀释标本,而金纳米-分子信标fqRT-PCR最低检出105 copies/μL的稀释标本。茎环引物fqRT-PCR与金纳米-分子信标fqRT-PCR检测miRNA-21比较,节约464个引物,节省时间208min。结论茎环引物fqRT-PCR检测miRNA具有快速、灵敏、重复性好,可用于临床定量分析miRNA。

Objective To establish a quantitative detection method of stem-loop primer based real time fluorescent quantitative reverse transcriptase-polymerase chain reaction（fqRT-PCR） for micro RNA（miRNA）.Methods 300 patients with myocardial infarction were collected to serve as myocardial infarction group,which subdivided into acute,subacute and chronic group according to their condition.100 healthy volunteers were selected as the control group in corresponding period.stem-loop primer based fqRT- PCR,gold nanoparticle-molecular beacon fqRT-PCR were used to detect serum miRNA-21 expression level.Results Serum miR- NA-21 expression level of patients in acute and chronic group were higher than those in subacute and control group（P〈0.05）. Serum miRNA-21 expression level of patients in subacute group was higher than that in control group（P〈0.05）.The minimum of 102 copies/μL diluted specimen was detected by stem-loop primer based fqRT-PCR while 105 copies/μL by gold nanoparticle-molecular beacon fqRT-PCR.Compared with gold nanoparticle-molecular beacon fqRT-PCR,stem-loop primer based fqRT-PCR re- trenched 464 primers and 208 min.Conclusion Stem-loop primers fqRT-PCR detection for miRNA is rapid,sensitive and reproduc- ible,which can be used in clinical quantitative analysis of miRNA.