title: Nanoliter droplet array for microRNA detection based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR.
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 1.5 × 10(5) to 1.5 × 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.
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Zeqi
authors.initials: Z
authors.lastname: Yu
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.forename: Yunxia
authors.initials: Y
authors.lastname: Zhang
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Qun
authors.initials: Q
authors.lastname: Fang
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Xi
authors.forename: Bo
authors.initials: B
authors.lastname: Yao
mesh_terms: D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D011088:DNA Ligases / Q000378:metabolism*; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism*; D003959:Diamines; D020869:Gene Expression Profiling; D055029:Inverted Repeat Sequences; D051379:Mice; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D036103:Nanotechnology / Q000295:instrumentation*; D009930:Organic Chemicals / Q000737:chemistry*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
chemical_list: D052160:Benzothiazoles; D015342:DNA Probes; D003959:Diamines; D035683:MicroRNAs; C544354:Mirn122 microRNA, mouse; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D011088:DNA Ligases