Shi Rui, Sun Ying-Husan, Zhang Xing-Hai, Chiang Vincent L
Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC, USA.
Methods Mol Biol. 2012;822:53-66. doi: 10.1007/978-1-61779-427-8_4.
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.
逆转录聚合酶链反应（RT-PCR）是分析RNA丰度的最重要技术之一。微小RNA（miRNA）是一组20至24个核苷酸的调节性小RNA，在植物和动物中发挥重要作用。然而，miRNA的小尺寸使得它们难以通过传统的RT-PCR技术进行检测和定量。在此，我们描述了一种专门设计用于定量miRNA的聚（T）接头RT-PCR方法。在该方法中，包括miRNA在内的总RNA通过使用聚（A）聚合酶和ATP的聚（A）加尾反应进行延伸。具有聚（A）尾的miRNA通过由聚（T）接头引发的逆转录转化为cDNA，然后使用miRNA特异性正向引物和通用聚（T）接头反向引物进行PCR扩增。RT-PCR扩增可以通过实时检测或终点检测来监测，以定量miRNA转录水平。PCR扩增子可以进行测序以验证特定miRNA基因的表达。
