摘要
目的:分析小鼠大脑组织microRNA(miRNA)表达谱,为研究特定miRNA及其靶基因、中枢神经系统miRNA相关的调节网络,以及miRNA在中枢神经系统疾病中的作用提供参考。方法:采用RNA加尾和引物延伸序列RT-PCR法,将精心设计的特异引物按Tm值排成阵列,用梯度实时PCR仪在不同的退火温度扩增每一个miRNA,并计算出每种miRNA相对于内参的表达量。结果:共检测了285个miRNA,阳性者260个,它们丰度不同,频数分布图大致呈正态。大部分miRNA的表达水平与已发表的芯片结果一致,而阳性率高于芯片结果。源自同一miRNA前体的2个成熟miRNA表达量,有些相差无几,有些则相差甚多。同簇相邻miRNA的丰度有些相近,有些则差异明显,提示miRNA转录后的归宿决定其丰度。结论:采用实时定量PCR阵列法,分析了小鼠大脑组织的miRNA表达谱,特别是获得了一些低丰度miRNA在小鼠大脑中表达的数据,这将有助于对这些低丰度miRNA的功能研究。
Objective：To examine global expression levels of microRNAs （miRNAs） in mouse cerebrum and to provide an important basis for detailed studies of individual miRNAs, their target genes, the miRNA-related regulatory networks in the mammalian central nervous system, and their implications in diseases. Methods： Low molecular weight RNA from cerebrum of five C57BL/6J mice were tailed and reverse transcribed by extended RT-primer. miRNA primers were carefully designed and arrayed on plates according to the Tm of each primer. PCR was carried out at different annealing temperatures using a gradient real-time PCR instrument. The relative expression level of each miRNA was calculated using 5sRNA for normalization. Results： Among the 285 miRNAs detected, 260 were positive with varying abundance. Their frequency distribution was approximately a normal distribution. The expression levels of most miRNAs were in accordance with previously published results by microarray. However, the positive rate was higher than that detected by microarray, miRNAs originating from the same hairpin precursors expressed at similar or significantly different levels. Clusters of proximal miRNAs were similar or quite different in abundance. It is suggested that the fate of miRNA after transcription determined their abundance. Conclusion： Using the RNA-tailing and primer-extension PCR array method, we obtained expression profile of miRNA in mouse cerebrum, especially the relative expression data of many low abundant miRNA in mouse cerebrum, which will be of special help for studying the fine-tuning function of low-level miRNAs.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2009年第2期152-157,共6页
Journal of Peking University:Health Sciences
基金
国家自然科学基金(30540033)资助~~
关键词
微RNAS
基因表达谱
逆转录聚合酶链反应
小鼠
MicroRNA
Gene expression profiling
Reverse transcriptase polymerase chain reaction
Mice
