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)

Anal Chim Acta. 2019 Oct 24:1077:208-215. doi: 10.1016/j.aca.2019.05.028. Epub 2019 May 16.

Authors

Mingcheng Xu¹, Jiawei Ye¹, Dan Yang¹, Abdu Ahmed Abdullah Al-Maskri¹, Haihong Hu¹, Cheulhee Jung², Sheng Cai³, Su Zeng⁴

Affiliations

¹ Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, Zhejiang University, Hangzhou, Zhejiang, 310058, China.
² Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea.
³ Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, Zhejiang University, Hangzhou, Zhejiang, 310058, China. Electronic address: caisheng@zju.edu.cn.
⁴ Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, Zhejiang University, Hangzhou, Zhejiang, 310058, China. Electronic address: zengsu@zju.edu.cn.

PMID: 31307711
DOI: 10.1016/j.aca.2019.05.028

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.

MeSH terms

Cell Line, Tumor
DNA / chemistry
DNA / genetics
DNA Probes / chemistry
DNA Probes / genetics
DNA-Directed DNA Polymerase / chemistry
Humans
Limit of Detection
MicroRNAs / analysis*
MicroRNAs / genetics
Nucleic Acid Hybridization
Polymerase Chain Reaction / methods*
Sensitivity and Specificity
Thermococcus / enzymology

Substances

DNA Probes
MIRN200 microRNA, human
MicroRNAs
DNA
Tli polymerase
DNA-Directed DNA Polymerase