Ultrasensitive detection of miRNA via one-step rolling circle-quantitative PCR (RC-qPCR)

Mingcheng Xu, Jiawei Ye, Dan Yang, Abdu Ahmed Abdullah AL-maskri, Haihong Hu, Cheulhee Jung, S. Cai, Su Zeng

    Research output: Contribution to journalArticlepeer-review

    36 Citations (Scopus)

    Abstract

    A novel microRNA (miRNA) quantification method has been developed using one-step rolling circle-quantitative PCR (RC-qPCR) analysis. Vent (exo-) DNA polymerase is firstly utilized to combine a rolling circle amplification (RCA) and qPCR in one step with high sensitivity and specificity in our RC-qPCR assay. Before performing the RC-qPCR, a padlock probe is ligated only when it is perfectly hybridized with miRNA. This ligation-based miRNA assay is highly specific for mature miRNAs, discriminating among related miRNAs that differ by as little as one nucleotide. It exhibits a dynamic range of seven orders of magnitude with a detection limit of 500 aM, and could be also used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs).

    Original languageEnglish
    Pages (from-to)208-215
    Number of pages8
    JournalAnalytica Chimica Acta
    Volume1077
    DOIs
    Publication statusPublished - 2019 Oct 24

    Bibliographical note

    Funding Information:
    We acknowledge financial support from the National Natural Science Foundation of China (Grant 81573389 ), the National Key R&D Program of China ( 2017YFC0908600 ), the Zhejiang Provincial Natural Science Foundation of China ( LY18H300003 ). Scientific Research Fund of Zhejiang Provincial Education Department ( Y201430444 ).

    Publisher Copyright:
    © 2019 Elsevier B.V.

    Keywords

    • MiR-200a
    • MiRNA detection
    • One-step amplification
    • Rolling circle amplification
    • Rolling circle-quantitative PCR (RC-qPCR)
    • Vent (exo-) DNA polymerase

    ASJC Scopus subject areas

    • Analytical Chemistry
    • Biochemistry
    • Environmental Chemistry
    • Spectroscopy

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