Chapter title |
Poly(T) adaptor rt-PCR.
|
---|---|
Chapter number | 4 |
Book title |
Next-Generation MicroRNA Expression Profiling Technology
|
Published in |
Methods in molecular biology, December 2011
|
DOI | 10.1007/978-1-61779-427-8_4 |
Pubmed ID | |
Book ISBNs |
978-1-61779-426-1, 978-1-61779-427-8
|
Authors |
Shi R, Sun YH, Zhang XH, Chiang VL, Rui Shi, Ying-Husan Sun, Xing-Hai Zhang, Vincent L. Chiang, Shi, Rui, Sun, Ying-Husan, Zhang, Xing-Hai, Chiang, Vincent L. |
Abstract |
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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