title: Dumbbell probe-mediated cascade isothermal amplification: a novel strategy for label-free detection of microRNAs and its application to real sample assay.
abstract: Considering the great significance of microRNAs (miRNAs) in cancer detection and typing, the development of sensitive, specific, quantitative, and low-cost methods for the assay of expression levels of miRNAs is desirable. We describe a highly efficient amplification platform for ultrasensitive analysis of miRNA (taking let-7a miRNA as a model analyte) based on a dumbbell probe-mediated cascade isothermal amplification (DP-CIA) strategy. The method relies on the circularization of dumbbell probe by binding target miRNA, followed by rolling circle amplification (RCA) reaction and an autonomous DNA machine performed by nicking/polymerization/displacement cycles that continuously produces single-stranded G-quadruplex to assemble with hemin to generate a color signal. In terms of the high sensitivity (as low as 1 zmol), wide dynamic range (covering 9 orders of magnitude), good specificity (even single-base difference) and easy operation (one probe and three enzymes), the proposed label-free assay is successfully applied to direct detection of let-7a miRNA in real sample (total RNA extracted from human lung tissue), demonstrating an attractive alternative for miRNA analysis for gene expression profiling and molecular diagnostics, particularly for early cancer diagnosis.
authors.affiliation: Key Laboratory of Biochemical Analysis, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China. bisai11@126.com
authors.forename: Sai
authors.initials: S
authors.lastname: Bi
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Cui
authors.forename: Li
authors.initials: L
authors.lastname: Li
mesh_terms: D015342:DNA Probes / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics; D035683:MicroRNAs / Q000032:analysis*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
chemical_list: D015342:DNA Probes; D035683:MicroRNAs; C487931:mirnlet7 microRNA, human; D004259:DNA-Directed DNA Polymerase