Developmet Of Nanoliter-Scale Droplet Array For Real Time Reverse Transcription Polvmerase Chain Reaction And Its Application In Single-Cell MicrpRNA Quantification

Amplification and quantitation of nucleic acids is essential for studies in gene expression and molecular diagnostics. It has become one of the most widely used techniques in the field of life science, and has brought revolutionary breakthrough in biology, environmental science, agronomy, medicine and other research fields. Recently, more and more efforts have been put into the miniaturization of PCR systems. Microfluidic PCR system, not only maintains the traditional advantages of PCR systems, such as high specificity and high sensitivity, but also brings many new advantages including low sample consumption, high throughput analysis, andsystem miniaturization and integration. Microfluidic PCR techniques will provide a promising platform for single-cell gene analysis and bring new possibilities for many important researches, including early diagnosis of cancers, prenatal diagnosis, forensic identification, and proteomic research.In Chapter1, the current progress and applications of microfluidic PCR systems are reviewed. Various microfluidic PCR systems are comprehensively summarized, described, and classified according to the structures of microfluidic reactors. The microfluidic PCR systems for single-cell gene analysis are introduced in details.In Chapter2, we developed a nanoliter-scale droplet-array system for performing parallel real-time quantitative RT-PCR (RT-qPCR) assays. The droplet array was generated on the chemically modified surface of silicon chip using a traditional pipette, and covered with a layer of mineral oil to eliminate evaporation during PCR cycling. Multiple reagent addition in droplets could be easily achieved using the pipette through the layer of oil. We demonstrated its feasibility to perform multistep qRT-PCR assays for microRNA quantification. Reliable and sensitive two step real time qRTPCR assay for microRNA measurement was performed within500nL droplets. It has a dynamic range spanning six orders of magnitude, and allows for quantification of microRNA input from103to109copies per reaction. We applied the present system for quantitative measurement of mir-122expression across five cultured cell lines. The minimum total RNA input was as low as1pg per assay, which showed its potential for gene quantification at single cell level. We envision the microfluidic droplet-array based RT-PCR system could be a universal and low-cost platform for gene quantification in routine biological laboratories.In Chapter3, we developed a nanoliter-scale and automated droplet array system for quantificating the expression of miRNA in single cells with two-step RT-qPCR assay. All the droplet manipulations and operations for single cell gene analysis, including droplet generation, cell encapsulation, cell lysis, reverse transcription of RNA, and real-time PCR detection, were fully integrated in the present system. To test the performance of real-time RT-qPCR assay using the system, synthetic microRNA-122over six orders (from30to106copies) of magnitude was measured in50-nL droplets, and excellent linear relationship between the logarithm of copy number of mir-122input and threshold cycle (Ct) value was obtained. The detect limit was30copies/droplet, which was much lower than that in conventional large-volume RT-PCR assays, demonstrating the high PCR amplification efficiency in nanoliter-scale droplets. We also comprehensively studied the inhibition effect of PBS from cell suspension on the PCR efficiency and detection limit, and an optimized PBS concentration of80μM was obtained. The present system was successfully applied to quantify the expression of mir-122in single Huh-7cells using an array of50-nL,256droplets to demonstrate its potential in single-cell analysis.