荧光定量PCR对微小RNA的检测及应用

MicroRNA's Detection with Fluorescence Quantitative PCR and Its Clinical Usage

目的:建立一种定量、简单、快速、敏感的微小RNA(miRNA,miR)检测方法,并用其观察一些病理状态下有关miRNA的变化.方法:通过特殊设计的茎环结构的反转录引物,对miRNA进行反转录,然后用相应miRNA的正反向引物对该miRNA进行定量检测,观察重复性和特异性.用此法检测了人巨细胞病毒(HCMV)对滋养细胞(HPT-8)细胞内miR-513表达水平的影响,也检测了结直肠癌组织miR-145水平.结果:用该法扩增miRNA特异性强,结果重复性好,该法检测到HCMV感染对miR-513表达的梯度效应.也发现癌组织和正常组织间miR-145的表达有明显的差异.结论:用荧光定量PCR可以对微小RNA快速有效的检测,在临床工作中可得到很好的应用.

Purpose： To establish a quantitative, sensitive, simple and rapid method for the detection of microRNA （miRNA, miR） and the use of this method in some clinical situations. Method：Take use of the hairpin structure of miRNA, we designed specialized primer for the reverse transcription of corresponding miRNA and amplified the miRNA by using real time PCR. We used this method in detecting miR-513 in human cytotrophoblast cell line - HPT-8 after human cytomegalovirus infection. We also detected miR-145 in colorectal cancer tissues. Results： With our method, fmiRNA could be specifically amplified and the result was repeatable. By using this method, we could observe miR-S13 change in HPT-8 cells after different titers human cytomegalovirus infection. We found also that miR-145 changed in colorectal tumors compared with normal colorectal tissues. （P 〈0.01）. Conclusion： Real time PCR can be a very effective method in detecting miRNA. This method has vast utilities in clinical practices.