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; Sheng Cai; Su Zeng

doi:10.1016/j.aca.2019.05.028

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, Sheng Cai, Su Zeng

Research output: Contribution to journal › Article › peer-review

14 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 language	English
Journal	Analytica Chimica Acta
DOIs	https://doi.org/10.1016/j.aca.2019.05.028
Publication status	Published - 2019 Jan 1

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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Cite this

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

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).",

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

author = "Mingcheng Xu and Jiawei Ye and Dan Yang and {Abdullah AL-maskri}, {Abdu Ahmed} and Haihong Hu and Cheulhee Jung and Sheng Cai and Su Zeng",

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AU - Xu, Mingcheng

AU - Ye, Jiawei

AU - Yang, Dan

AU - Abdullah AL-maskri, Abdu Ahmed

AU - Hu, Haihong

AU - Jung, Cheulhee

AU - Cai, Sheng

AU - Zeng, Su

PY - 2019/1/1

Y1 - 2019/1/1

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KW - MiR-200a

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KW - One-step amplification

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KW - Rolling circle-quantitative PCR (RC-qPCR)

KW - Vent (exo-) DNA polymerase

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