5 years ago

Target-initiated labeling for the dual-amplified detection of multiple microRNAs

Target-initiated labeling for the dual-amplified detection of multiple microRNAs
Herein we exploited a novel target-initiated labeling strategy for the multiplexed detection of microRNAs (miRNAs) by coupling duplex-specific nuclease (DSN) with terminal deoxynucleotidyl transferase (TdT). In the presence of target miRNA, the immobilized and 3′-blocked capture probes hybridized with target and thus the formed DNA-RNA hybrid was recognized by DSN. DSN mediated the digestion of 3′-phosphated capture probes (CPs) in the hybrids and synchronously target was released and recycled for another round of hybridization and cleavage. The cleaved CP fragments with a free 3′-OH were then elongated and labeled with multiple biotin-dUTP nucleotides by TdT. Fluorescence reporter streptavidin-phycoerythin was finally added to react with the immobilized biotins and render fluorescence signals. This dual-amplification labeling strategy was successfully demonstrated to sensitively detect multiple miRNAs, taking advantage of DSN-mediated target recycling and TdT-catalyzed multiple signal modification with analysis by a commercial Luminex xMAP array platform. Our experimental results showed the simultaneous quantitative measurement of three sequence-specific miRNAs at concentrations from 1 pM to 2.5 nM. Attempts were also made to directly detect miRNAs in total RNA extracted from cancer cells. The dual-amplification labeling strategy reported here shows a great potential for the development of a method for the multiplexed, sensitive, selective, and simple analysis of multiple miRNAs in tissues or cells for biomedical research and clinical early diagnosis.

Publisher URL: www.sciencedirect.com/science

DOI: S0003267017309662

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.