Quantitative stem-loop RT-PCR for detection of microRNAs

Varkonyi-Gasic, E. & Hellens, R.P. (2011) Quantitative stem-loop RT-PCR for detection of microRNAs. Methods in Molecular Biology, 744, pp. 145-157.

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Abstract

Plant microRNAs (miRNAs) are a class of endogenous small RNAs that are essential for plant development and survival. They arise from larger precursor RNAs with a characteristic hairpin structure and regulate gene activity by targeting mRNA transcripts for cleavage or translational repression. Efficient and reliable detection and quantification of miRNA expression has become an essential step in understanding their specific roles. The expression levels of miRNAs can vary dramatically between samples and they often escape detection by conventional technologies such as cloning, northern hybridization and microarray analysis. The stem-loop RT-PCR method described here is designed to detect and quantify mature miRNAs in a fast, specific, accurate and reliable manner. First, a miRNA-specific stem-loop RT primer is hybridized to the miRNA and then reverse transcribed. Next, the RT product is amplified and monitored in real time using a miRNA-specific forward primer and the universal reverse primer. This method enables miRNA expression profiling from as little as 10 pg of total RNA and is suitable for high-throughput miRNA expression analysis.

Impact and interest:

50 citations in Scopus
54 citations in Web of Science®
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ID Code: 71614
Item Type: Journal Article
Refereed: Yes
Additional Information: Book Title:
RNAi and Plant Gene Function Analysis;
Book Subtitle:
Methods and Protocols
Additional URLs:
DOI: 10.1007/978-1-61779-123-9_10
ISSN: 1940-6029
Divisions: Current > QUT Faculties and Divisions > Science & Engineering Faculty
Deposited On: 15 May 2014 03:21
Last Modified: 20 May 2014 06:13

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