Journal
TALANTA
Volume 85, Issue 4, Pages 1760-1765Publisher
ELSEVIER
DOI: 10.1016/j.talanta.2011.06.075
Keywords
microRNA; Droplet array; Real-time PCR; Ligation; SYBR Green
Categories
Funding
- National Natural Science Foundation of China [20905064, 20890020]
- Research Fund for the Doctoral Program of Higher Education of China [20090101120007]
- Zhejiang Provincial Natural Science Foundation of China [Y409002]
- Chinese Universities Scientific Fund [KYJD09001]
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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 1.5 x 10(5) to 1.5 x 10(10) copies). Linear relationship of the standard curve (R-2 = 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. (C) 2011 Elsevier B.V. All rights reserved.
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