Selective miRNA quantitation with high-temperature thermal gel electrophoresis

MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression and correlate to the prognosis of numerous diseases. To support research efforts elucidating the roles of miRNAs in pathogenesis, rapid and inexpensive analytical methods are required to quantify miRNAs from biological samples. The challenge of developing new analyses with these time and cost constraints is compounded by the short sequence lengths and high degrees of homology between miRNAs that hinder detection selectivity. This report describes the development of a high-temperature thermal gel electrophoresis (TGE) method to rapidly quantify miRNAs with single-nucleotide resolution using low-cost microfluidic devices. Fluorescent probes were designed for three miRNAs that differed in sequence by one or two nucleotides. A microfluidic analysis was optimized to enrich miRNA-probe hybrids into a high-concentration band and then automatically initiate a separation to resolve each species. Analyses conducted at 30 °C exhibited significant off-target hybridization, as the different-yet-structurally-similar miRNAs bound to each probe, which biased measurements. To overcome this problem, the stability of thermal gels at elevated temperatures was exploited to conduct analyses. At 50 °C, off-target hybrids melted to prevent their detection without impeding the enrichment or separation of on-target hybrids. Selectivity studies validated that high-temperature TGE prevented off-target hybrids from interfering with the quantitative responses of the target miRNAs. This work demonstrates that TGE affords rapid, highly selective analyses of structurally similar miRNAs in low-complexity microfluidic devices, which is expected to facilitate diverse biomedical research.