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

TLDR

MicroRNAs are small non‑coding RNAs essential for development and environmental responses, yet current gel‑based assays lack throughput, sensitivity, and specificity, making reliable detection crucial. The study presents RT‑PCR protocols for detecting and quantifying miRNAs, including an alternative assay that enhances specificity for low‑abundance targets. The methods employ end‑point and real‑time looped RT‑PCR, plus an alternative real‑time PCR assay designed to improve specificity for low‑abundant miRNAs. The assays detect miRNAs from as little as 20 pg of plant RNA or 0.1 µl of phloem sap, reveal differential expression between phloem sap and surrounding vascular tissue, and enable fast, sensitive, specific, high‑throughput miRNA profiling.

Abstract

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 μl 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.