Protocol : A highly sensitive RT-PCR method for detection and quantification of microRNAs

Varkonyi-Gasic, Erika, Wu, Rongmei, Wood, Marion, Walton, Eric F., & Hellens, Roger P. (2007) Protocol : A highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods, 3(12).

View at publisher (open access)


MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 l of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

Impact and interest:

357 citations in Scopus
Search Google Scholar™
310 citations in Web of Science®

Citation counts are sourced monthly from Scopus and Web of Science® citation databases.

These databases contain citations from different subsets of available publications and different time periods and thus the citation count from each is usually different. Some works are not in either database and no count is displayed. Scopus includes citations from articles published in 1996 onwards, and Web of Science® generally from 1980 onwards.

Citations counts from the Google Scholar™ indexing service can be viewed at the linked Google Scholar™ search.

Full-text downloads:

76 since deposited on 13 May 2014
38 in the past twelve months

Full-text downloads displays the total number of times this work’s files (e.g., a PDF) have been downloaded from QUT ePrints as well as the number of downloads in the previous 365 days. The count includes downloads for all files if a work has more than one.

ID Code: 71449
Item Type: Journal Article
Refereed: Yes
Additional Information: Cited By (since 1996):182
Export Date: 6 May 2014
Source: Scopus
Art. No.: 12
Additional URLs:
DOI: 10.1186/1746-4811-3-12
ISSN: 1746-4811
Divisions: Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 2007 Varkonyi-Gasic et al; licensee BioMed Central Ltd.
Copyright Statement: This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Deposited On: 13 May 2014 00:38
Last Modified: 16 Jun 2014 06:41

Export: EndNote | Dublin Core | BibTeX

Repository Staff Only: item control page