Development of a sensitive primer extension method for direct detection and quantification of miRNAs from plants.

Abstract

MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate target gene expression in various organisms. Functional studies are therefore required to determine their temporal and spatial expression patterns. Primer extension has been used as a sensitive and reliable approach to identify miRNAs (∼21–22 nt) in the mammalian system and can be used in other systems such as plants. However, a well-defined method is required for ease of application and reproducibility. Here, a radioactive primer extension method was developed for the quantitative detection of miRNAs found in total RNA samples from plants. As a proof of concept, miR173 and miR828 were detected by primer extension in total RNA samples isolated from Arabidopsis. The assay involved the extension reaction of the miRNA guide strand with a radiolabeled specific primer. Using a manual DNA sequencer, primers extended with reverse transcriptase were separated on a denaturing polyacrylamide gel. The gel was then dried and exposed to a PhosphorImager screen for size-dependent product identification up to a single base difference. Quantification was done based on the intensity of radioactive signals by normalizing the cDNA products to an internal control. The primer extension was proven to be efficient to detect and quantify miRNAs in plant total RNA samples without subsequent enrichment of low-molecular-weight RNA species. This method, optimized for Arabidopsis, can be applied to a wide variety of organisms for the detection and quantification of miRNAs as well as siRNAs.