SYBR green实时荧光定量PCR检测微小RNA21的方法建立及在食管鳞癌中的应用

A Method for Detection miR-21 by using SYBR Green Real-time Fluorescent Quantitative PCR

目的:建立SYBR green实时荧光定量PCR检测微小RNA miR-21的技术平台及应用。方法:设计微小RNA21和U6的的颈环结构反转录引物和PCR扩增引物,以U6为内参利用SYBR green实时荧光定量PCR法检测小鼠各器官中的微小RNA21的含量。提取16例食管鳞癌患者的肿瘤组织及其近旁组织中的总RNA,检测其微小RNA21表达水平。结果:SYBR green实时荧光定量PCR检测U6和微小RNA21含量的熔解曲线单一,PCR产物特异。在Balb/c小鼠的4种器官中,肝脏、脾脏、肾脏分别为脑组织的8.71、5.38、3.47倍。16对食管鳞癌患者的样本中,14例微小RNA21的拷贝数高于其近旁组织约10.58倍(p<0.01)。结论:此研究成功建立了SYBR green荧光定量PCR法检测小鼠和人微小RNA-21含量的技术平台,为进一步阐述miR-21在食管鳞癌的发生中的作用提供了新方向。

Objective：To establish a metbod of SYBR green real - time quantitative PCR for detecting levels of miR -21 and apply in detection. Method： According to the sequence of miR -21 and U6, the stem -loop reverse transcriptional pr/mer, the primers of real - time PCR were designed and synthesized. The expression levels of miR -21 in different organs of mouse was measured by SYBR green real- time quantitative PCR to detect the accuracy of tiffs method with U6 as control. In addition, sixteen specimens of cancer and corresponding normal tissues were collected from sixteen esophageal squamous cancer patients. The total RNA were extracted and reverse transcriptied. The expression levels of miR -21 were confirmed. Result： The melting curve of miR -21 analysis in four organs of Balb/c mouse showed a single peak, the product of PCR is specific. The quantitative PCR results showed that, miR -21 in brain levels is less than hver, spleen and kidney. And compared with the control samples, miR -21 levels in the esophageal cancer samples was significantly higher about 10.58 fold（p 〈 0.01 ）. Conclusion ： The method of detecting miR - 21 levels by SYBR green real - time quantitative PCR was established successfidly both in mouse and human samples. Moreover, the present result provided a new view between miR-21 and esophageal cancer carcinogenesis. w