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Linear-Hairpin Variable Primer RT-qPCR for MicroRNA

Abstract

Here, we present a highly specific, sensitive and cost-effective system to quantify microRNAs (miRNAs) expression based on two-step RT-qPCR with EvaGreen detection chemistry, called linear-hairpin variable primer RT-qPCR. It takes advantage of the novel designed variable primer, which is initially designed to be linear, extending to form a hairpin structure and replacing the target miRNA for cyclic RT. Then the RT product is quantified by conventional EvaGreen based qPCR. The results show that this method has a dynamic range of 8 logs and the sensitivity sufficient to directly detect down to 4 target miRNA molecules with a total analysis time less than 2 hours. It is capable to discriminate between similar miRNAs, leading to accurate representation of the mature miRNA content in a sample. The RT step can be multiplexed and the 8 miRNA profiles measured in 7 mouse tissues by this method show excellent correlation with the commercial standard TaqMan RT-qPCR assays (r2=0.9881).

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Publication details

The article was received on 17 Oct 2018, accepted on 02 Dec 2018 and first published on 04 Dec 2018


Article type: Edge Article
DOI: 10.1039/C8SC04621B
Citation: Chem. Sci., 2019, Accepted Manuscript
  • Open access: Creative Commons BY-NC license
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    Linear-Hairpin Variable Primer RT-qPCR for MicroRNA

    L. Lan, Q. Guo, H. Nie, C. Zhou, Q. Cai, J. Huang and X. Meng, Chem. Sci., 2019, Accepted Manuscript , DOI: 10.1039/C8SC04621B

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