Triple cascade reactions: An ultrasensitive and specific single tube strategy enabling isothermal analysis of microRNA at sub-attomole level

Biosens Bioelectron. 2016 Jun 15:80:378-384. doi: 10.1016/j.bios.2016.01.059. Epub 2016 Jan 24.

Abstract

Sensitive and specific analysis of microRNAs (miRNAs) in a single tube without the need of thermal cycler instrument would greatly facilitate the investigation of miRNA-associated regulatory circuits and diseases. Homogeneous isothermal amplification assays are attractive in conducting single tube assays that can minimize contamination-prone steps and simplifies assay procedures. However, the relative low amplification efficiency and high detection background remain as bottlenecks restricting their more versatile applications. In this work, we have developed a novel isothermal exponential enzymatic amplification (IEEA) strategy for miRNAs analysis. By rational triple cascade amplification cycles of target recycling, nicking-replication reaction, and DNAzyme catalysis, the strategy exhibited high signal amplification efficiency (10(4)-10(9) folds of amplification in 1h) with very low detection background and excellent specificity. As a result, the miR-27a target model was rapidly determined with a limit of detection down to 0.79 aM (S/N=3), corresponding to 94 copies of the miRNA molecule in a 200 μL sample solution. The levels of miR-27a in atherosclerotic sprague-dawley rats were accurately quantified. The strategy is anticipated to have an important impact on the development of simple and rapid molecular diagnostic applications for any short oligonucleotides.

Keywords: Atherosclerotic sprague-dawley rats; Homogeneous isothermal amplification assays; Isothermal exponential enzymatic amplification (IEEA) strategy; MicroRNAs analysis.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Atherosclerosis / genetics*
  • Biosensing Techniques / methods
  • Limit of Detection
  • Male
  • MicroRNAs / analysis*
  • MicroRNAs / genetics
  • Nucleic Acid Amplification Techniques / methods*
  • Rats, Sprague-Dawley

Substances

  • MIRN27 microRNA, rat
  • MicroRNAs