Nanoliter Droplet Array for Quantification of MicroRNA by Two-Step Real Time Reverse Transcription Polymerase Chain Reaction

Zhang Yunxia; Zhu Ying; Yao Bo; Fang Qun

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Nanoliter Droplet Array for Quantification of MicroRNA by Two-Step Real Time Reverse Transcription Polymerase Chain Reaction

Zhang Yunxia 1 #,Zhu Ying 2,Yao Bo 2 * #,Fang Qun 1

1. Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University

2.Department of Chemistry, Zhejiang University

*Correspondence author

#Submitted by

Subject:

Funding: Research Fund for the Doctoral Program of Higher Education of China （No.20090101120007）

Opened online:19 July 2010

Accepted by: none

Citation: Zhang Yunxia,Zhu Ying,Yao Bo.Nanoliter Droplet Array for Quantification of MicroRNA by Two-Step Real Time Reverse Transcription Polymerase Chain Reaction[OL]. [19 July 2010] http://en.paper.edu.cn/en_releasepaper/content/4379033

In the past decade, great effort has been put into the miniaturization of genetic tests such as PCR, however, current parallel micro-PCR devices including droplet based PCR chip and emulsion PCR system are usually designed for performing multiple PCR reactions in identical conditions with the same primers and templates, thus limiting their application for singleplex assays. In this paper, we developed a 6×6 nanoliter droplet array to perform reliable and parallel real time RT-PCR which is generally applied for quantitative biology. Multi-step reagent addition and parallel PCR assay could be simply performed on this droplet array with a traditional pipette. The total reaction volume is only 500nL which is reduced more than 70-fold compared to conventional RT-PCR assay. The total RNA input was as low as 3pg per reaction, which showed a great potential for gene quantification at single cell level. We successfully applied the platform to quantitative measurement of mir-122 in five cultured cell lines and this droplet based PCR chip would be a universal platform for not only for microRNA and other small non-coding RNA, also for mRNA or even DNA quantification.

Keywords:Analytical Chemistry; droplet array; real time PCR; microRNA

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