Rapid quantification of microRNA-375 through one-pot primer-generating rolling circle amplification

A recent surge of interest in microRNA has been driven by its discovery as a circulating biomarker of disease, with many diagnostic test platforms currently under development. Alternatives to widely used microRNA quantification methods such as quantitative reverse transcriptase PCR (qRT-PCR) are needed for use in portable and point-of-care devices which are incompatible with complex sample processing workflows and thermal cycling. Rolling circle amplification (RCA) is a one-pot assay technique which directly amplifies nucleic acids using sequence-specific microRNA priming to initiate a single-step isothermal reaction that is compatible with simple devices. Sensitivity remains a limitation of RCA methods, however, and detection limits do not typically reach the femtomolar level in which microRNA targets are present in blood. RCA assays have previously been improved by digestion of the amplification products using a nicking endonuclease to exponentially generate new reaction primers. Here we describe how a ligation-free version of this technique performed in a single tube can be used to improve the limit of detection for microRNA-375, an important blood biomarker for prostate cancer. Endonuclease addition changes a linear process into an exponential amplification reaction which results in a 61-fold improvement of the limit of detection (5.9 fM), a dynamic range wider than 5-log(10), and a shorter reaction time. By eliminating the need for microRNA reverse transcription and thermal cycling, this single-step one-pot method provides a more rapid and simplified alternative to qRT-PCR for ultrasensitive microRNA quantification in blood extracts.