title: Signal amplification of microRNAs with modified strand displacement-based cycling probe technology.
abstract: Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA. The products generated by SDA are captured by a molecular beacon (MB), which is designed to initiate the second amplification cycle, with a similar principle to the cycling probe technology (CPT), which is based on repeated digestion of the DNA-RNA hybrid by the RNase H. After one sample enrichment and two steps of signal amplification, 0.1 pM of let-7a can be detected. The miRNA assay exhibits a great dynamic range of over 100 orders of magnitude and high specificity to clearly discriminate a single base difference in miRNA sequences. This isothermal amplification does not require any special temperature control instrument. The assay is also about signal amplification rather than template amplification, therefore minimising contamination issues. In addition, there is no need for the reverse transcription (RT) process. Thus the amplification is suitable for miRNA detection.
authors.affiliation: Department of Pharmacology, Jinling Hospital, State Key Laboratory of Analytical Chemistry for Life Science, Medical School of Nanjing University, Nanjing 210002, China. ghzhou@nju.edu.cn and Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China. songqinxin@cpu.edu.cn.
authors.forename: Huning
authors.initials: H
authors.lastname: Jia
authors.affiliation: Huadong Research Institute for Medicine and Biotechnics, Nanjing, 210002, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Bu
authors.affiliation: Department of Pharmacology, Jinling Hospital, State Key Laboratory of Analytical Chemistry for Life Science, Medical School of Nanjing University, Nanjing 210002, China. ghzhou@nju.edu.cn.
authors.forename: Bingjie
authors.initials: B
authors.lastname: Zou
authors.affiliation: Department of Pharmacology, Jinling Hospital, State Key Laboratory of Analytical Chemistry for Life Science, Medical School of Nanjing University, Nanjing 210002, China. ghzhou@nju.edu.cn.
authors.forename: Jianping
authors.initials: J
authors.lastname: Wang
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand.
authors.forename: Shalen
authors.initials: S
authors.lastname: Kumar
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand.
authors.forename: Janet L
authors.initials: JL
authors.lastname: Pitman
authors.affiliation: Department of Pharmacology, Jinling Hospital, State Key Laboratory of Analytical Chemistry for Life Science, Medical School of Nanjing University, Nanjing 210002, China. ghzhou@nju.edu.cn.
authors.forename: Guohua
authors.initials: G
authors.lastname: Zhou
authors.affiliation: Department of Pharmacology, Jinling Hospital, State Key Laboratory of Analytical Chemistry for Life Science, Medical School of Nanjing University, Nanjing 210002, China. ghzhou@nju.edu.cn and Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China. songqinxin@cpu.edu.cn.
authors.forename: Qinxin
authors.initials: Q
authors.lastname: Song
mesh_terms: D015342:DNA Probes; D035683:MicroRNAs / Q000032:analysis*; D021141:Nucleic Acid Amplification Techniques; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D035683:MicroRNAs