A fishhook probe-based rolling circle amplification (FP-RCA) assay for efficient isolation and detection of microRNA without total RNA extraction

Abstract

MicroRNA (miRNA) analysis has vital significance as a potential biomarker in clinical diagnosis and cancer research. In this study, a simple and practical technique was proposed for the detection of microRNAs from cell lysates based on fishhook probe-mediated rolling circle amplification (RCA) and fluorescence imaging with a smartphone. Compared with reported methods related to miRNA detection, this method mainly focused on simplicity, low cost and portability. Fishhook probes were designed and immobilized on the surface of streptavidin-coated magnetic beads for effectively recognizing and capturing target miRNAs, thus achieving simple and selective separation from the sample matrix. Moreover, the captured miRNAs initiated and transferred RCA reaction into solution, thus making the heterogeneous separation and homogeneous amplification reactions compatible. Excess circular probes as well as other nucleic acids were removed by two-step magnetic separation, minimizing nonspecific amplification and background signal. Using magnetic separation, high specificity was obtained even for one base mismatch strand. Moreover, the detection of miR-21 in cell lysates was performed without total RNA extraction. The fishhook probe-based rolling circle amplification (FP-RCA) assay integrated isolation and detection of miRNAs into a compact process, which was simple and effective without the need for bulky and expensive equipment such as centrifuge, thermal cycler and fluorescent microscope except for a blue light source device and a smartphone camera. Our study may provide a low-cost and reliable platform for miRNA detection and related research.