Sensitive, Multiplexed Electrophoretic miRNA Detection Using Peptide Nucleic Acids in Micelle-Elfse

miRNAs are short, noncoding nucleic acids that are biomarkers for a growing number of disease states, including breast cancer, Alzheimer's disease, and heart disease. Because they are only 20-22 bases in length, and are present in trace quantities, they are difficult to detect by conventional PCR and array methods. Here, we present an electrophoretic detection system that uses synthetic γ-substituted peptide nucleic acids, which bind miRNA with great specificity and stability. Low detection limits are achieved using an ultrabright probe in the form of a DNA oligomer saturated with intercalating dye. The micelle-ELFSE method has been extended to kilobase-length DNA to provide ultrabright fluorophores for sub-attomole detection of miRNA in less than 3 minutes. The use of surfactant running buffers, rather than gels, makes the method compatible with high amounts of serum (10-100 mg/ml) with minimal peak distortion. Finally, the use of gamma-substituted peptide nucleic acid probes gives great binding stability and specificity, allowing for miRNA detection in a sandwich format. The method can easily distinguish members of the closely related let-7 series. We will discuss efforts to concentrate miRNA by ITP-based sample stacking as well as theoretical limits of peak capacity for multiplexed detection in capillary and microchip electrophoresis. We will also discuss applications in early-stage cancer screening and other medical applications of this rapid, selective miRNA detection scheme.