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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Acknowledgments
This work was supported by research grants from the US Department of Energy Division of Energy Biosciences DE-FG02-03ER15442 and the National Science Foundation, Plant Genome Research Program DBI-0922391 to VLC, and from the North Carolina State University Forest Biotechnology Industrial Research Consortium (FORBIRC) to VLC and RS. X-HZ wishes to thank the Chiang Laboratory for hosting his sabbatical leave.
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Shi, R., Sun, YH., Zhang, XH., Chiang, V.L. (2012). Poly(T) Adaptor RT-PCR. In: Fan, JB. (eds) Next-Generation MicroRNA Expression Profiling Technology. Methods in Molecular Biology, vol 822. Humana Press. https://doi.org/10.1007/978-1-61779-427-8_4
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DOI: https://doi.org/10.1007/978-1-61779-427-8_4
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