| Chapter title |
Stem-Loop qRT-PCR for the Detection of Plant microRNAs.
|
|---|---|
| Chapter number | 13 |
| Book title |
Plant Epigenetics
|
| Published in |
Methods in molecular biology, January 2017
|
| DOI | 10.1007/978-1-4899-7708-3_13 |
| Pubmed ID | |
| Book ISBNs |
978-1-4899-7706-9, 978-1-4899-7708-3
|
| Authors |
Erika Varkonyi-Gasic |
| Editors |
Igor Kovalchuk |
| Abstract |
Plant microRNAs (miRNAs) play important roles in the posttranscriptional regulation of protein-coding genes, and they are essential for a normal development and survival. Mature miRNAs are cleaved from larger precursor RNAs and are typically 21-22 nt long.The small size, the lack of a common feature like a poly(A) tail, 3' end-modifications, and presence of a precursor-all these factors affect the detection and hinder the quantification of miRNAs. The stem-loop qRT-PCR method described here is designed to detect and quantify mature miRNAs in a fast, specific, accurate, and reliable manner. Firstly, a miRNA-specific stem-loop RT primer is hybridized to miRNA and then reverse transcribed. Next, the RT product is amplified and monitored in real time using a miRNA-specific forward primer and a universal reverse primer. This method enables miRNA expression profiling from as little as 10 pg of total RNA, and it is suitable for a relatively high-throughput analysis of miRNA expression. |
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