Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10559454 | Talanta | 2011 | 6 Pages |
Abstract
In this paper, a nanoliter droplet array based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR for quantification of microRNA was developed. By employing T4 RNA ligase 2 instead of T4 DNA ligase, we designed simplified stem-loop probes to perform microRNA-templated DNA ligation and reduced the non-specific ligation of T4 DNA ligase. SYBR green I dye was employed instead of TaqMan probes in present miniaturized real-time PCR systems. Specifically, we optimized the dosage of SYBR Green I dye in nanoliter droplet and verified the performance of this system by detecting synthetic mir-122 with a 6 logs dynamic range (from 1.5Â ÃÂ 105 to 1.5Â ÃÂ 1010 copies). Linear relationship of the standard curve (R2Â =Â 0.9997) and high PCR amplification efficiency (96.83%) were obtained under the optimized conditions. We detected the expression of mir-122 across five mouse tissues and the result was consistent with that TaqMan microRNA assay. We think this miniaturized real-time PCR platform reduced the detection cost considerably, thus showing the great potential to quantitative biology.
Related Topics
Physical Sciences and Engineering
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Authors
Zeqi Yu, Ying Zhu, Yunxia Zhang, Juan Li, Qun Fang, Jianzhong Xi, Bo Yao,