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 language | English |
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Pages (from-to) | 208-215 |
Number of pages | 8 |
Journal | Analytica Chimica Acta |
Volume | 1077 |
DOIs | |
Publication status | Published - 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