Enzymatic Ligation-based Real-time PCR On Microchip

In recent years, real-time PCR based on microfluidic chip has been paid more and more attention on account of high throughput, low cost and low reagent consumption. This technique has been widely applied in many fields of life science. Herein, we demonstrated a nanoliter droplet array based on SYBR Green for real-time quantitative PCR, and developed a method of PCR base on enzymatic stem-loop probes ligation to quantify the expression of microRNA (miRNAs) in biological samples. T4RNA ligase2, employed instead of T4DNA ligase in this method, significantly diminished the influence of non-specific ligation and improved the specificity of detection. By employing SYBR Green fluorescent dye in droplet-based miniaturized real-time PCR system, operating cost was reduced considerably.In Chapter1, the principle and major application of traditional PCR and fluorescenct real-time quantitative PCR are introduced. The methods of fluorescence labeling are described detailedly. Different types of microchip-based PCR and their advantage are reviewed. Furthermore, various techniques of detecting rm’RNA are discussed comprehensively.In chapter2, a nanoliter droplet array based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR for quantification of miRNA was developed. Ligase-mediated gene detection could overcome the weakness of reverse transcription. By employing T4RNA. ligase2instead of T4DNA ligase, we designed simplified stem-loop probes to perform miRNA-templated DNA ligation and reduced the non-specific ligation of T4DNA ligase. SYBR Green I dye was employed instead of TaqMan probes, which could reduce detecting cost considerably. Specifically, we optimized the dosage of SYBR Green I dye in500nL droplet and verified the performance of this system by detecting synthetic mir-122with a6logs dynamic range (from1.5×105to1.5×1010copies). Linear relationship of the standard curve (R2=0.9997) and high PCR amplification efficiency (96.83%) were obtained under the optimized conditions. We detected the expression of mir-122across five mouse tissues (spleen, kidney, liver, lung and brain) and the result was consistent with that TaqMan microRNA assay.We think this miniaturized real-time PCR platform could become a simply, sensitive, accurate and especially low-cost tool for detecting miRNA, and had great potential to large-scale application.