Highly sensitive multiplex detection of microRNA by competitive DNA strand displacement fluorescence assay

MicroRNA (miRNA) is a small non-coding RNA with the size of 18-22 nucleotide. MiRNAs play a major role in the gene expression and regulation. They influence more than 50% of protein coding genes in mammalian genome which regulate many cellular functions. Their dysregulation can lead to cancer and other diseases. MiRNAs have been shown to be associated with breast cancers. Previously q-PCR based assays have been used successfully for detection and quantification of miRNAs, however, these assays are expensive and cumbersome. Here, we report the detection of few breast cancer microRNA sequences using fluorescence displacement assay. The assay employed a fluorophore-quencher pair by hybridization of fluorescently labelled cDNA of miRNA and a quencher labelled short DNA. Upon hybridization, the fluorophore and the quencher become in close vicinity to each other leading to significant fluorescence quenching. In the presence of target miRNA, the fluorophore and quencher are separated due to the duplex dissociation and form stable cDNA-miRNA double strand which leads to increase in the fluorescence intensity. The assay has dynamic range from 0.2 to 250 nM miRNA concentration. We could detect miRNA as low as 1 pM level. The method was applied to detect the miRNA spiked to the total RNA extracted from whole blood. The fluorescence based method has been validated using standard Q-PCR method. This could prove a promising method for the detection of miRNAs.