RNA加尾和引物延伸RT-PCR法实时定量检测microRNA

目的:用改进的RNA加尾和引物延伸RT-PCR法实时定量检测组织中的microRNAs(miRNAs),并评价其敏感性和特异性.方法:提取组织中的小于200 bp的RNA,用poly(A)聚合酶在其3'末端加尾,再用5'带40 nt延伸序列的单碱基锚定Olig-dT引物进行反转录,得到约80 nt的cDNA,然后用特异引物进行SYBR Green实时定量PCR扩增.结果:该法线性范围宽,可检测低丰度的miRNAs,能特异区分3'末端碱基不同的亚型,但对序列中间个别碱基不同的亚型不能鉴别.对15种miRNA在大鼠心、肝和大脑皮层的相对表达的检测验证了该法的可靠性.另外,海马组织中的miRNAs表达与大脑皮层组织有一定差异.熔解曲线的单一峰型和电泳检测到的单一条带都证明了扩增的特异性.结论:该法可快速、敏感地检测不同组织中大多数miRNAs的表达差异,可用于寻找组织特异及疾病相关的miRNAs.

Real-time quantification of microRNAs by RNA-tailing and primer-extension RT-PCR

OBJECTIVE: To optimize and evaluate the modified RNA-tailing and primer-extension RT-PCR method in relative quantification of microRNAs (miRNAs) in several kinds of tissues. METHODS: Small-sized RNAs (<200 bp) were extracted and polyadenylated by poly(A) polymerase. One-base anchored oligo-dT primers with 40 nt extension at their 5'-ends were used to reversely transcribe the poly(A)-tailed miRNAs. miRNAs were then amplified by SYBR Green real-time PCR using miRNA specific primers. RESULTS: This method had a high dynamic range and could detect low abundant miRNAs. The assay was capable of discriminating between related miRNA family members that differed by subtle sequence differences at 3' ends, but it could not distinguish those differed in the middle of their sequences. Fifteen mature miRNAs were differentially expressed in rat heart, liver, and cortex,which were in accordance with published results by other methods. The miRNA expression signature in hippocampus was different from that in cortex. miR-122a expression was higher and miR-124a expression was lower in hippocampus than in cortex. A single dissociation peak on the thermal melting curve and a single DNA band about 80 bp on 3.5% NuSeive agarose gel signified the amplification specificity for these miRNAs. CONCLUSION: This method enables fast and sensitive relative quantification for most miRNAs and can identify potential biomarkers specific to tissues or diseases.