New method for microRNA detection based on multimerization

Detection of specific microRNA (miRNA) is of great demand due to their essential role in genes regulation, stress response and development of diseases. However, mature miRNAs are small molecules that make it difficult to use routine amplification-based methods. Here, we report an approach for detection of miRNA based on a new type of isothermal amplification, namely, multimerization. The proposed technique is simple and versatile, excludes a reverse transcription step, and requires two conventional primers only and no additional stem-loop or fluorogenic probes. Only mature miRNAs can initiate multimerization, thereby, pri- or pre-miRNA are excluded from analysis, ensuring high accuracy of the assay. The approach was approved on miRNA from common wheat Triticum aestivum; the increase of Tae-miRNA159 level for plants affected by Stagonospora nodorum Berk infection was demonstrated. The obtained results allow to perform quantitative analysis, providing determination of specific targets with high reliability (detection limit of about 20 pM). [Display omitted] •Nucleic acids multimerization (MM) allows miRNA detection with high specificity.•Definite size of miRNA determines efficient generation of structures providing MM.•Only mature miRNA initiate MM, while precursors don't involve into reaction.•The proposed technique requires no reverse transcription step.•MM allows accurate quantification of specific miRNA targets.