放大器
底漆(化妆品)
逆转录酶
小RNA
生物
互补DNA
分子生物学
逆转录聚合酶链式反应
计算生物学
实时聚合酶链反应
聚合酶链反应
信号转导衔接蛋白
核糖核酸
cDNA末端的快速扩增
信使核糖核酸
基因
遗传学
化学
分子克隆
有机化学
作者
Rui Shi,Ying-Hsuan Sun,Xing‐Hai Zhang,Vincent L. Chiang
出处
期刊:Methods in molecular biology
日期:2011-12-01
卷期号:: 53-66
被引量:33
标识
DOI:10.1007/978-1-61779-427-8_4
摘要
Reverse transcription PCR (RT-PCR) is one of the most important techniques for analyzing RNA abundance. MicroRNAs (miRNAs) are a group of 20- to 24-nucleotide regulatory small RNAs which play an important role in plants and animals. However, the small size of miRNAs makes them difficult to be detected and quantified by conventional RT-PCR techniques. Here, we describe a poly(T) adaptor RT-PCR method specifically designed for quantifying miRNAs. In this method, total RNAs, including miRNAs, are extended by a poly(A) tailing reaction using poly(A) polymerase and ATP. The miRNA with a poly(A) tail is converted into cDNA through reverse transcription primed by a poly(T) adaptor, and then PCR-amplified using a miRNA-specific forward primer and a universal poly(T) adaptor reverse primer. The RT-PCR amplification can be monitored by real-time detection or by end-point detection for quantifying the miRNA transcript level. The PCR amplicons can be sequenced for validating the expression of the specific miRNA gene.
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