Reverse transcription-free digital-quantitative-PCR for microRNA analysis

MicroRNAs (miRNAs) are non-coding RNA sequences that regulate many biological processes and have become central targets of biomedical research. However, their naturally low abundances in biological samples necessitates the development of sensitive analytical techniques to conduct routine miRNA measurements in research laboratories. Digital PCR has the potential to meet this need because of its single-molecule detection capabilities, but PCR analyses of miRNAs are slowed by the ligation and reverse transcription steps first required to prepare samples. This report describes the development of a method to rapidly quantify miRNA in digital microwell arrays using base-stacking digital-quantitative-PCR (BS-dqPCR). BS-dqPCR expedites miRNA measurements by eliminating the need for ligation and reverse transcription steps, which reduces the time and cost compared to conventional miRNA PCR analyses. Under standard PCR thermocycling conditions, digital signals from miRNA samples were lower than expected, while signals from blanks were high. Therefore, a novel asymmetric thermocycling program was developed that maximized on-target signal from miRNA while minimizing non-specific amplification. The analytical response of BS-dqPCR was then evaluated over a range of miRNA concentrations. The digital PCR dimension increased in signal with increasing miRNA copy numbers. When the digital signal saturated, the quantitative PCR dimension readily discerned miRNA copy number differences. Overall, BS-dqPCR provides rapid, high-sensitivity measurements of miRNA over a wide dynamic range, which demonstrates its utility for routine miRNA analyses.