臺灣博碩士論文加值系統

在進行單細胞的研究過程中，分離出單細胞並且鑑定是不可或缺的步驟，但往往需付出大量的時間及金錢成本來達到此目的。本研究的目標在使用一個全新的平台系統，PanelChip™ Analysis System，建立方法使其能夠結合分離單細胞並收集的功能，同時完成單細胞基因圖譜鑑定；此系統包含微陣列晶片(2500格)與數位即時聚合酶鏈鎖反應平台的系統，與標準的即時聚合酶鏈鎖反應平台進行比較，以驗証數位即時聚合酶鏈鎖反應平台的效能展現。
在數位即時聚合酶鏈鎖反應平台系統與標準的即時聚合酶鏈鎖反應平台上進行標準品(pUC19)的連續稀釋實驗以驗証動態範圍及再現性。並在兩種平台使用兩種不同已知濃度的A549肺癌細胞樣本，測試其vimentin, E-cadherin, N-cadherin 與GAPDH的基因表現。更進一步的使用採血試管中常使用的抗擬血劑同時也是酶鏈鎖反應抑制劑-肝素(heparin)，作為兩種不同平台系統對於酶鏈鎖反應抑制劑的耐受性評估及比較。最後，提出單細胞分散的數學模型並且驗證了單細胞的分離方法，並且再進一步地將A549肺癌細胞以藥物(TGFβ1)誘導使其進行上皮間質轉化後進行單細胞基因圖譜分析檢測。
經過上列實驗得出的結論是，使用Panel Chip™ Analysis System進行數位即時聚合酶鏈鎖反應，在濃度範圍3.4至3.4 x10^8copies/μL具有良好的線性關係(R^2=0.9974)；並且不管是在2pg/μL樣品濃度( 變異系數1.97)或200pg/μL樣品濃度( 變異系數1.30)，都小於標準qPCR在2pg/μL樣品濃度( 變異系數3.94)或200pg/μL樣品濃度( 變異系數8.11)，代表著數位即時聚合酶鏈鎖反應方法的再現性優於傳統即時聚合酶鏈鎖反應方法；在酶抑制劑耐受性方面，數位即時聚合酶鏈鎖反應方法(IC50: 0.02IU/mL)亦大於標準即時聚合酶鏈鎖反應方法(IC50: 0.002IU/mL)，代表即時聚合酶鏈鎖反應方法對於肝素的耐受性優於標準即時聚合酶鏈鎖反應方法。經由實驗驗証，可以使用PanelChip™ Analysis System快速得到高通量的單細胞基因表現圖譜(約1000 單細胞/晶片)。數位即時聚合酶鏈鎖反應在臨床診斷和單細胞應用上具有發展的潛力。

In a single-cell study, isolating and identifying single cells are essential, but these processes often require a large investment of time or money. The aim of this study was to isolate and analyse single cells using a novel platform, the PanelChip™ Analysis System, which includes 2500 microwells chip and a digital real-time polymerase chain reaction (dqPCR) assay, in comparison with a standard RT-PCR (qPCR) assay.
Through the serial dilution of a known concentration standard, namely pUC19, the accuracy and sensitivity levels of two methodologies were compared. The two systems were also tested on the basis of expression levels of the genetic markers vimentin, E-cadherin, N-cadherin and GAPDH in A549 lung carcinoma cells at two known concentrations. Furthermore, the influence of a known PCR inhibitor commonly found in blood samples, heparin, was evaluated in both methodologies.
Finally, mathematical models were proposed and separation method of single cells was verified; moreover, gene expression levels during epithelial–mesenchymal transition in single cells under TGFβ1 treatment were measured.
The drawn conclusion is that dqPCR performed using PanelChip™ has good linearity (R^2=0.9974) at 3.4 to 3.4 x 10^8 copies/μL, and it is superior to the standard qPCR in terms of, reproducibility (at 2pg/μL, dqPCR CV:1.97 < qPCR CV:3.94 ; at 200pg/μL , dqPCR CV:1.30 < qPCR CV:8.11) and heparin tolerance (dqPCR IC50: 0.02IU/mL > qPCR IC50: 0.002IU/mL). The dqPCR assay is a potential tool for clinical diagnosis and single-cell applications.

口試委員會審定書 I
致謝 II
摘要 III
ABSTRACT V
TABLE OF CONTENTS VII
LIST OF TABLES XI
LIST OF FIGURES XII
CHAPTER 1 INTRODUCTION 1
1.1. Background 1
1.2. Purposes 2
1.3. Experimental Design 3
CHAPTER 2 LITERATURE REVIEW 5
2.1.Precision Medicine 5
2.1.1. What is Precision Medicine ? 5
2.1.2. W1hy Precision Medicine Important ? 6
2.2. Tumor Metastasis 7
2.2.1. Metastasis 7
2.2.2. Circulating Tumor Cells, CTCs 8
2.2.3. Epithelial-Mesenchymal Transition, EMT 9
2.3. Single-Cell Study 10
2.3.1.Why Single-Cell Important ? 10
2.3.2. Single-Cell Isolation 11
2.3.3. Single-Cell Analysis Instrument on the Market 12
2.4. Digital PCR 13
CHAPTER 3 MATERIALS AND METHODS 15
3.1. Biomaterial Preparation 15
3.1.1. Cell Preparation 15
3.1.2.Isolation of A549 Total RNA 15
3.1.3. pUC19 Standard DNA and Primer Pairs 15
3.2. Amplification Methods 16
3.2.1.RT-qPCR and qPCR 16
3.2.2. RT-dqPCR and dqPCR 16
3.3. Linearity and Sensitivity 18
3.4. Precision and Repeatability 18
3.5. Inhibitor Resistance 19
3.6. Cellular Distribution Across Microwell chip 19
3.7. Gene Expression Profiling of Epithelial-Mesenchymal Transformation in Single-Cells 19
3.8. Data Analysis 20
3.8.1. Dynamic Range of dqPCR 20
3.8.2. Evaluation of the Inhibitory Influenece of Heparin 21
3.8.3. Cellular Distribution in a Chip 22
3.8.4. Single-Cell Gene Expression Profiling 24
CHAPTER 4 RESULTS 26
4.1. Performance of PanelChipTM Analysis System 26
4.1.1. Serial Dilution of pUC19 Plasmd DNA Standard 26
4.1.2. Precision and Repeatability 28
4.1.3. Inhibitor Resistance 29
4.2. Cell Distribution Across the 2500 Microwell Chip 31
4.2.1. Cell distribution at Various Input Numbers 31
4.2.2. Epithelial-Mesenchymal Gene Expression Profiling in Single-Cells 32
CHAPTER 5 DISCUSSIONS 34
5.1. Performance of PanelChipTM Analysis System 34
5.1.1 Measuring the Linear Dynamic Range of dqPCR and qPCR system 34
5.1.2 Evaluate the Reproducibility of dqPCR and qPCR system 35
5.1.3 Determine the Heparin Tolerance of dqPCR and qPCR system 36
5.2.Single-Cell Analysis using PanelChipTM Analysis System 38
5.2.1. Using the Poisson Distribution to Obtain Single-Cell 38
5.2.2. Single-Cell Gene Expression Profiling of A549 cell treated with TGFβ1 39
CHAPTER 6 CONCLUSIONS 40
REFERENCE 41
TABLES 47
FIGURES 51

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